Therapeutic nanoparticles comprising a therapeutic agent and methods of making and using the same

ABSTRACT

The present disclosure generally relates to nanoparticles comprising a substantially hydrophobic acid and a therapeutic agent (1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea), or pharmaceutically acceptable salts thereof, and a polymer. Other aspects include methods of making and using such nanoparticles.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application61/953,628, filed on Mar. 14, 2014, which is incorporated by referenceherein in its entirety.

BACKGROUND

Systems that deliver certain drugs to a patient (e.g., targeted to aparticular tissue or cell type or targeted to a specific diseased tissuebut not normal tissue) or that control release of drugs have long beenrecognized as beneficial.

For example, therapeutics that include an active drug and that are,e.g., targeted to a particular tissue or cell type or targeted to aspecific diseased tissue but not to normal tissue, may reduce the amountof the drug in tissues of the body that are not targeted. This isparticularly important when treating a condition such as cancer where itis desirable that a cytotoxic dose of the drug is delivered to cancercells without killing the surrounding non-cancerous tissue. Effectivedrug targeting may reduce the undesirable and sometimes life threateningside effects common in anticancer therapy. In addition, suchtherapeutics may allow drugs to reach certain tissues they wouldotherwise be unable to reach.

Therapeutics that offer controlled release and/or targeted therapy alsomust be able to deliver an effective amount of drug, which is a knownlimitation in other nanoparticle delivery systems. For example, it canbe a challenge to prepare nanoparticle systems that have an appropriateamount of drug associated with each nanoparticle, while keeping the sizeof the nanoparticles small enough to have advantageous deliveryproperties.

Therapeutic agents containing at least one basic nitrogen atom (i.e.,protonatable nitrogen-containing therapeutic agents) represent animportant group of therapeutic agents. However, nanoparticleformulations of this class of drugs are often hindered by undesirableproperties, e.g., unfavorable burst release profiles and poor drugloading.

Accordingly, a need exists for nanoparticle therapeutics and methods ofmaking such nanoparticles that are capable of delivering therapeuticlevels of protonatable nitrogen-containing therapeutic agents to treatdiseases such as cancer, while also reducing patient side effects.

SUMMARY

The present invention relates to a therapeutic nanoparticle of thetherapeutic drug,1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor pharmaceutically acceptable salts thereof. More specifically, thepresent invention relates to a therapeutic nanoparticle comprising1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor pharmaceutically acceptable salts thereof and further comprising asubstantially hydrophobic acid. Additionally, the present inventionrelates to a therapeutic nanoparticle comprising1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor pharmaceutically acceptable salts thereof, a substantiallyhydrophobic acid, and a polymer selected from diblock poly(lactic)acid-poly(ethylene)glycol copolymer, a diblock poly(lacticacid-co-glycolic acid)-poly(ethylene)glycol copolymer and combinationthereof, wherein the therapeutic nanoparticle comprises about 10 toabout 30 weight percent poly(ethylene)glycol. The present invention alsorelates to a pharmaceutical composition comprising such nanoparticles,including a plurality of such nanoparticles, and a pharmaceuticallyacceptable excipient. In addition, the present invention relates to atherapeutic nanoparticle comprising about 0.05 to about 30 weightpercent of a substantially hydrophobic acid, about 0.2 to about 25weight percent of1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor a pharmaceutically acceptable salt thereof and about 50 to about99.75 weight percent of a copolymer selected from diblock poly(lactic)acid-poly(ethylene)glycol copolymer, a diblock poly(lacticacid-co-glycolic acid)-poly(ethylene)glycol copolymer and combinationthereof, wherein the therapeutic nanoparticle comprises about 10 toabout 30 weight percent poly(ethylene)glycol, as well as apharmaceutical composition comprising the therapeutic nanoparticle and aand a pharmaceutically acceptable excipient. The present invention alsorelates to a therapeutic nanoparticle comprising about 0.05 to about 30weight percent of a substantially hydrophobic acid, about 0.2 to about20 weight percent of1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor a pharmaceutically acceptable salt thereof and about 50 to about99.75 weight percent of a copolymer selected from diblock poly(lactic)acid-poly(ethylene)glycol copolymer, a diblock poly(lacticacid-co-glycolic acid)-poly(ethylene)glycol copolymer and combinationthereof, wherein the therapeutic nanoparticle comprises about 10 toabout 30 weight percent poly(ethylene)glycol, as well as apharmaceutical composition comprising the therapeutic nanoparticle and aand a pharmaceutically acceptable excipient.

Described herein are polymeric nanoparticles that include thetherapeutic agent,1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor a pharmaceutically acceptable salt thereof. This compound is basicand it is a protonatable nitrogen-containing therapeutic agent, asdefined herein below. Also described herein are methods of making andusing such therapeutic nanoparticles.

In one aspect, a therapeutic nanoparticle is provided. In this aspect,the therapeutic nanoparticle comprises about 0.05 to about 30 weightpercent of a substantially hydrophobic acid, about 0.2 to about 25weight percent of1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor a pharmaceutically acceptable salt thereof, wherein the pK_(a) of theprotonated form of the therapeutic agent is at least about 1.0 pK_(a)units greater than the pK_(a) of the hydrophobic acid, and about 50 toabout 99.75 weight percent of a copolymer selected from diblockpoly(lactic) acid-poly(ethylene)glycol copolymer, a diblock poly(lacticacid-co-glycolic acid)-poly(ethylene)glycol copolymer and combinationthereof, wherein the therapeutic nanoparticle comprises about 10 toabout 30 weight percent poly(ethylene)glycol. In one embodiment, thetherapeutic nanoparticle comprises about 0.05 to about 30 weight percentof a substantially hydrophobic acid, about 0.2 to about 20 weightpercent of1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor a pharmaceutically acceptable salt thereof, wherein the pK_(a) of theprotonated form of the therapeutic agent is at least about 1.0 pK_(a)units greater than the pK_(a) of the hydrophobic acid, and about 50 toabout 99.75 weight percent of a copolymer selected from diblockpoly(lactic) acid-poly(ethylene)glycol copolymer, a diblock poly(lacticacid-co-glycolic acid)-poly(ethylene)glycol copolymer and combinationthereof, wherein the therapeutic nanoparticle comprises about 10 toabout 30 weight percent poly(ethylene)glycol.

In certain embodiments, the therapeutic nanoparticle comprises1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand PLA-PEG (in a 16:5 molar ratio) in a weight ratio of about 1:7(therapeutic agent:PLA-PEG). In certain embodiments, the therapeuticnanoparticle comprises1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand PLA-PEG (in a 16:5 molar ratio) in a weight ratio of about 1:5(therapeutic agent:PLA-PEG). In certain embodiments, the therapeuticnanoparticle comprises1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand PLA-PEG (in a 16:5 molar ratio) in a weight ratio of about 1:4(therapeutic agent:PLA-PEG). In certain embodiments, the therapeuticnanoparticle comprises1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand PLA-PEG (in a 16:5 molar ratio) in a weight ratio of about 1:14(therapeutic agent:PLA-PEG). In certain embodiments, the therapeuticnanoparticle comprises1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand PLA-PEG (in a 16:5 molar ratio) in a weight ratio of about 1:3(therapeutic agent:PLA-PEG).

In another aspect, the therapeutic nanoparticle comprises asubstantially hydrophobic acid, wherein the molar ratio of thesubstantially hydrophobic acid to the aforementioned therapeutic agentranges from about 0.25:1 to about 2:1, about 0.2 to about 25 weightpercent of the aforementioned therapeutic agent, wherein the pK_(a) ofthe protonated therapeutic agent is at least about 1.0 pK_(a) unitsgreater than the pK_(a) of the hydrophobic acid, and about 50 to about99.75 weight percent of a polymer selected from diblock poly(lactic)acid-poly(ethylene)glycol copolymer, diblock poly(lacticacid-co-glycolic acid)-poly(ethylene)glycol copolymer and combinationthereof, wherein the therapeutic nanoparticle comprises about 10 toabout 30 weight percent poly(ethylene)glycol. In one embodiment, thetherapeutic nanoparticle comprises a substantially hydrophobic acid,wherein the molar ratio of the substantially hydrophobic acid to theaforementioned therapeutic agent ranges from about 0.25:1 to about 2:1,about 0.2 to about 20 weight percent of the aforementioned therapeuticagent, wherein the pK_(a) of the protonated therapeutic agent is atleast about 1.0 pK_(a) units greater than the pK_(a) of the hydrophobicacid, and about 50 to about 99.75 weight percent of a polymer selectedfrom diblock poly(lactic) acid-poly(ethylene)glycol copolymer, diblockpoly(lactic acid-co-glycolic acid)-poly(ethylene)glycol copolymer andcombination thereof, wherein the therapeutic nanoparticle comprisesabout 10 to about 30 weight percent poly(ethylene)glycol.

In certain embodiments, the therapeutic nanoparticle comprises asubstantially hydrophobic acid and the aforementioned therapeutic agent,wherein the pK_(a) of the protonated therapeutic agent is at least about1.0 pK_(a) units greater than the pK_(a) of the hydrophobic acid, and apolymer selected from diblock poly(lactic) acid-poly(ethylene)glycolcopolymer or a diblock poly(lactic acid-co-glycolicacid)-poly(ethylene)glycol copolymer and combination thereof.

In some embodiments, the therapeutic nanoparticle comprises theaforementioned therapeutic agent, a substantially hydrophobic acid,wherein the molar ratio of the substantially hydrophobic acid to thetherapeutic agent ranges from about 0.25:1 to about 2:1 and wherein thepK_(a) of the protonated therapeutic agent is at least about 1.0 pK_(a)units greater than the pK_(a) of the hydrophobic acid, and a polymerselected from diblock poly(lactic) acid-poly(ethylene)glycol copolymeror a diblock poly(lactic acid-co-glycolic acid)-poly(ethylene)glycolcopolymer and combination thereof.

In some embodiments, the molar ratio of the substantially hydrophobicacid to the aforementioned therapeutic agent is about 0.5:1 to about1.5:1. In certain embodiments, the molar ratio of the substantiallyhydrophobic acid to the aforementioned therapeutic agent is about 0.75:1to about 1.25:1. In certain embodiments, the molar ratio of thesubstantially hydrophobic acid to the aforementioned therapeutic agentis about 0.25:1 to about 1:1. In certain embodiments, the pK_(a) of theprotonated form of the aforementioned therapeutic agent is at leastabout 2.0 pK_(a) units greater than the pK_(a) of the hydrophobic acid.In other embodiments, the pK_(a) of the protonated form of theaforementioned therapeutic agent is at least about 4.0 pK_(a) unitsgreater than the pK_(a) of the hydrophobic acid.

In another aspect, a the therapeutic nanoparticle comprises ahydrophobic ion-pair comprising a hydrophobic acid and theaforementioned therapeutic agent; wherein the difference between thepK_(a) of the protonated form of the aforementioned therapeutic agentand the hydrophobic acid is at least about 1.0 pK_(a) units, and about50 to about 99.75 weight percent of a diblock poly(lactic)acid-poly(ethylene)glycol copolymer, wherein the poly(lactic)acid-poly(ethylene)glycol copolymer has a number average molecularweight of about 15 kDa to about 20 kDa poly(lactic acid) and a numberaverage molecular weight of about 4 kDa to about 6 kDapoly(ethylene)glycol. In certain embodiments of this aspect of theinvention, the difference between the pK_(a) of the protonated form ofthe aforementioned therapeutic agent and the hydrophobic acid is atleast about 2.0 pK_(a) units. In other embodiments, the differencebetween the pK_(a) of the protonated form of the aforementionedtherapeutic agent and the hydrophobic acid is at least about 4.0 pK_(a)units.

In certain embodiments, the therapeutic nanoparticle comprises about0.05 to about 20 weight percent of the hydrophobic acid.

In some embodiments, the substantially hydrophobic acid has a log P ofabout 2 to about 8, where P is the octanol/water partition coefficientof the hydrophobic acid. In some embodiments, the substantiallyhydrophobic acid has a log P of about 4 to about 8. In some embodiments,the substantially hydrophobic acid has a log P of about 2 to about 7.

In some embodiments, the substantially hydrophobic acid has a pK_(a) inwater of about −1.0 to about 5.0. In other embodiments, thesubstantially hydrophobic acid has a pK_(a) in water of about 2.0 toabout 5.0.

In certain embodiments, the substantially hydrophobic acid and theaforementioned therapeutic agent form a hydrophobic ion pair in thetherapeutic nanoparticle.

In some embodiments, the hydrophobic acid is a fatty acid. For example,in certain embodiments, the fatty acid is a saturated fatty acid,including, but not limited to, caproic acid, enanthic acid, caprylicacid, pelargonic acid, capric acid, undecanoic acid, lauric acid,tridecylic acid, myristic acid, pentadecylic acid, palmitic acid,margaric acid, stearic acid, nonadecylic acid, arachidic acid,heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid,pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid,nonacosylic acid, melissic acid, henatriacontylic acid, lacceroic acid,psyllic acid, geddic acid, ceroplastic acid, hexatriacontylic acid, orcombinations thereof. In other embodiments, the fatty acid is an omega-3fatty acid, including, but not limited to, hexadecatrienoic acid,alpha-linolenic acid, stearidonic acid, eicosatrienoic acid,eicosatetraenoic acid, eicosapentaenoic acid, heneicosapentaenoic acid,docosapentaenoic acid, docosahexaenoic acid, tetracosapentaenoic acid,tetracosahexaenoic acid, or combinations thereof. In still otherembodiments, the fatty acid is an omega-6 fatty acid, including, but notlimited to, linoleic acid, gamma-linolenic acid, eicosadienoic acid,dihomo-gamma-linolenic acid, arachidonic acid, docosadienoic acid,adrenic acid, docosapentaenoic acid, tetracosatetraenoic acid,tetracosapentaenoic acid, or combinations thereof. In certain otherembodiments, the fatty acid is an omega-9 fatty acid, including, but notlimited to, oleic acid, eicosenoic acid, mead acid, erucic acid,nervonic acid, or combinations thereof. In other embodiments, the fattyacid is a polyunsaturated fatty acid, including, but not limited,rumenic acid, α-calendic acid, β-calendic acid, jacaric acid,α-eleostearic acid, β-eleostearic acid, catalpic acid, punicic acid,rumelenic acid, α-parinaric acid, β-parinaric acid, bosseopentaenoicacid, pinolenic acid, podocarpic acid, or combinations thereof.

In certain embodiments, the hydrophobic acid is a bile acid. Forexample, in some embodiments, the bile acid includes but is not limitedto, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid,hycholic acid, beta-muricholic acid, cholic acid, lithocholic acid, anamino acid-conjugated bile acid, or combinations thereof. In someembodiments, the bile acid is cholic acid. In other embodiments, theamino acid-conjugated bile acid is a glycine-conjugated bile acid or ataurine-conjugated bile acid.

In certain embodiments, the hydrophobic acid includes but is not limitedto, dioctyl sulfosuccinic acid, 1-hydroxy-2-naphthoic acid,dodecylsulfuric acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, pamoic acid, undecanoic acid, orcombinations thereof.

In other embodiments, the hydrophobic acid has a molecular weight ofbetween about 200 Da and about 800 Da.

In certain embodiments, the hydrophobic acid is pamoic acid. In otherembodiments, the hydrophobic acid is oleic acid. In some embodiments,the weight ratio of1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureato oleic acid is about 6:1. In some embodiments, the weight ratio of1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureato ratio to pamoic acid is about 1.8:1.

In some embodiments, the therapeutic nanoparticle comprises about 1 toabout 20 weight percent of the aforementioned therapeutic agent. Inother embodiments, the therapeutic nanoparticle comprises about 1 toabout 15 weight percent of the aforementioned therapeutic agent. Inother embodiments, the therapeutic nanoparticle comprises about 2 toabout 20 weight percent of the afore-mentioned therapeutic agent. Inother embodiments, the therapeutic nanoparticle comprises about 2 toabout 15 weight percent of the afore-mentioned therapeutic agent. Instill other embodiments, the therapeutic nanoparticle of comprises about4 to about 20 weight percent of the aforementioned therapeutic agent. Instill other embodiments, the therapeutic nanoparticle of comprises about4 to about 15 weight percent of the aforementioned therapeutic agent. Incertain other embodiments, the therapeutic nanoparticle comprises about5 to about 20 weight percent of the aforementioned therapeutic agent. Incertain other embodiments, the therapeutic nanoparticle comprises about5 to about 10 weight percent of the aforementioned therapeutic agent.

In some embodiments, the therapeutic nanoparticle substantially retainsthe therapeutic agent for at least 1 minute when placed in a phosphatebuffer solution at 37° C. In certain embodiments, the therapeuticnanoparticle substantially immediately releases less than about 30% ofthe therapeutic agent when placed in a phosphate buffer solution at 37°C. In certain other embodiments, the therapeutic nanoparticle releasesabout 10 to about 45% of the therapeutic agent over about 1 hour whenplaced in a phosphate buffer solution at 37° C. In some embodiments, thetherapeutic nanoparticle releases about 0.01 to about 15% of thetherapeutic agent over about 4 hours when placed in a phosphate buffersolution at 37° C. In some embodiments, the therapeutic nanoparticlereleases about 0.01 to about 15% of the therapeutic agent over about 10hours when placed in a phosphate buffer solution at 37° C. In someembodiments, the therapeutic nanoparticle releases about 0.01 to about25% of the therapeutic agent over about 20 hours when placed in aphosphate buffer solution at 37° C. In some embodiments, the therapeuticnanoparticle releases about 1 to about 40% of the therapeutic agent overabout 40 hours when placed in a phosphate buffer solution at 37° C. Instill other embodiments, the therapeutic nanoparticle has a releaseprofile that is substantially the same as a release profile for acontrol nanoparticle that is substantially the same as the therapeuticnanoparticle except that it does not contain a fatty acid or bile acid.

In certain embodiments, the poly(lactic) acid-poly(ethylene)glycolcopolymer has a poly(lactic) acid number average molecular weightfraction of about 0.6 to about 0.95. In certain other embodiments, thepoly(lactic) acid-poly(ethylene)glycol copolymer has a poly(lactic) acidnumber average molecular weight fraction of about 0.6 to about 0.8. Instill other embodiments, the poly(lactic) acid-poly(ethylene)glycolcopolymer has a poly(lactic) acid number average molecular weightfraction of about 0.75 to about 0.85. In other embodiments, thepoly(lactic) acid-poly(ethylene)glycol copolymer has a poly(lactic) acidnumber average molecular weight fraction of about 0.7 to about 0.9.

In certain embodiments, the therapeutic nanoparticle comprises about 10to about 25 weight percent poly(ethylene)glycol. In certain otherembodiments, the therapeutic nanoparticle comprises about 10 to about 20weight percent poly(ethylene)glycol. In still other embodiments, thetherapeutic nanoparticle comprises about 15 to about 25 weight percentpoly(ethylene)glycol. In other embodiments, the therapeutic nanoparticlecomprises about 20 to about 30 weight percent poly(ethylene)glycol.

In certain embodiments, the poly(lactic) acid-poly(ethylene)glycolcopolymer has a number average molecular weight of about 15 kDa to about20 kDa poly(lactic acid) and a number average molecular weight of about4 kDa to about 6 kDa poly(ethylene)glycol.

In some embodiments, the therapeutic nanoparticle further comprisesabout 0.2 to about 30 weight percent poly(lactic)acid-poly(ethylene)glycol copolymer functionalized with a targetingligand. In other embodiments, the therapeutic nanoparticle furthercomprises about 0.2 to about 30 weight percent poly(lactic)acid-co-poly(glycolic) acid-poly(ethylene)glycol copolymerfunctionalized with a targeting ligand. In certain embodiments, thetargeting ligand is covalently bound to the poly(ethylene)glycol.

In certain embodiments, the hydrophobic acid is a polyelectrolyte. Forexample, in some embodiments, the polyelectrolyte includes but is notlimited to poly(styrene sulfonic acid), polyacrylic acid,polymethacrylic acid, or combinations thereof.

In certain embodiments, a contemplated therapeutic nanoparticle furthercomprises a mixture of two or more substantially hydrophobic acids. Forexample, in some embodiments, a contemplated therapeutic nanoparticlecomprises a mixture of two substantially hydrophobic acids, a mixture ofthree substantially hydrophobic acids, a mixture of four substantiallyhydrophobic acids, or a mixture of five substantially hydrophobic acids.In some embodiments, the mixture of substantially hydrophobic acidscomprises oleic acid and cholic acid. In other embodiments, the mixtureof two substantially hydrophobic acids are oleic acid and cholic acid.

In another aspect, the therapeutic nanoparticle is prepared byemulsification of a first organic phase comprising a first polymer, theaforementioned therapeutic agent, and a substantially hydrophobic acid,thereby forming an emulsion phase; quenching of the emulsion phase,thereby forming a quenched phase, and finally filtering of the quenchedphase to recover the therapeutic nanoparticles.

In some embodiments, the hydrophobic acid used in preparing thetherapeutic nanoparticle is a fatty acid. For example, in certainembodiments, the fatty acid used in preparing the therapeuticnanoparticle is a saturated fatty acid including, but not limited to,caproic acid, enanthic acid, caprylic acid, pelargonic acid, capricacid, undecanoic acid, lauric acid, tridecylic acid, myristic acid,pentadecylic acid, palmitic acid, margaric acid, stearic acid,nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid,tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid,heptacosylic acid, montanic acid, nonacosylic acid, melissic acid,henatriacontylic acid, lacceroic acid, psyllic acid, geddic acid,ceroplastic acid, hexatriacontylic acid, or combinations thereof. Inother embodiments, the fatty acid used in preparing the therapeuticnanoparticle is an omega-3 fatty acid including, but not limited to,hexadecatrienoic acid, alpha-linolenic acid, stearidonic acid,eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid,heneicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid,tetracosapentaenoic acid, tetracosahexaenoic acid, or combinationsthereof. In still other embodiments, the fatty acid used in preparingthe therapeutic nanoparticle is an omega-6 fatty acid including, but notlimited to, linoleic acid, gamma-linolenic acid, eicosadienoic acid,dihomo-gamma-linolenic acid, arachidonic acid, docosadienoic acid,adrenic acid, docosapentaenoic acid, tetracosatetraenoic acid,tetracosapentaenoic acid, or combinations thereof. In certain otherembodiments, the fatty acid used in preparing the therapeuticnanoparticle is an omega-9 fatty acid including, but not limited to,oleic acid, eicosenoic acid, mead acid, erucic acid, nervonic acid, orcombinations thereof. In other embodiments, the fatty acid used inpreparing the therapeutic nanoparticle is a polyunsaturated fatty acidincluding, but not limited to, rumenic acid, α-calendic acid, β-calendicacid, jacaric acid, α-eleostearic acid, β-eleostearic acid, catalpicacid, punicic acid, rumelenic acid, α-parinaric acid, β-parinaric acid,bosseopentaenoic acid, pinolenic acid, podocarpic acid, or combinationsthereof.

In certain embodiments, the hydrophobic acid used in preparing thetherapeutic nanoparticle is a bile acid including, but not limited to,chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, hycholicacid, beta-muricholic acid, cholic acid, lithocholic acid, an aminoacid-conjugated bile acid, or combinations thereof. In some embodiments,the bile acid is cholic acid. In other embodiments, the aminoacid-conjugated bile acid is a glycine-conjugated bile acid or ataurine-conjugated bile acid.

In certain embodiments, the hydrophobic acid used in preparing thetherapeutic nanoparticle includes, but is not limited to, dioctylsulfosuccinic acid, 1-hydroxy-2-naphthoic acid, dodecylsulfuric acid,naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, pamoicacid, undecanoic acid, or combinations thereof.

In other embodiments, the hydrophobic acid used in preparing thetherapeutic nanoparticle has a molecular weight of between about 200 Daand about 800 Da.

In certain embodiments, the hydrophobic acid used in preparing thetherapeutic nanoparticle is pamoic acid. In other embodiments, thehydrophobic acid used in preparing the therapeutic nanoparticle is oleicacid. In some embodiments, the weight ratio of1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureato oleic acid used in preparing the therapeutic nanoparticle is about6:1. In some embodiments, the weight ratio of1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureato ratio to pamoic acid used in preparing the therapeutic nanoparticleis about 1.8:1. In some embodiments, the weight ratio of1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureato oleic acid used in preparing the therapeutic nanoparticle is 6:1. Insome embodiments, the weight ratio of1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureato ratio to pamoic acid used in preparing the therapeutic nanoparticleis 1.8:1.

In certain embodiments, the first polymer used in preparing thetherapeutic nanoparticle is poly(lactic) acid-poly(ethylene)glycolcopolymer. In other embodiments, the first polymer is poly(lactic)acid-co-poly(glycolic) acid-poly(ethylene)glycol copolymer. In certainembodiments, the poly(lactic) acid-poly(ethylene)glycol copolymer has apoly(lactic) acid number average molecular weight fraction of about 0.6to about 0.95. In certain other embodiments, the poly(lactic)acid-poly(ethylene)glycol copolymer has a poly(lactic) acid numberaverage molecular weight fraction of about 0.6 to about 0.8. In stillother embodiments, the poly(lactic) acid-poly(ethylene)glycol copolymerhas a poly(lactic) acid number average molecular weight fraction ofabout 0.75 to about 0.85. In other embodiments, the poly(lactic)acid-poly(ethylene)glycol copolymer has a poly(lactic) acid numberaverage molecular weight fraction of about 0.7 to about 0.9.

In certain embodiments, the therapeutic nanoparticle is prepared usingabout 10 to about 25 weight percent poly(ethylene)glycol. In certainother embodiments, about 10 to about 20 weight percentpoly(ethylene)glycol is used. In still other embodiments, about 15 toabout 25 weight percent poly(ethylene)glycol is used. In otherembodiments, about 20 to about 30 weight percent poly(ethylene)glycol isused.

In certain embodiments, the poly(lactic) acid-poly(ethylene)glycolcopolymer used in preparing the therapeutic nanoparticle has a numberaverage molecular weight of about 15 kDa to about 20 kDa poly(lacticacid) and a number average molecular weight of about 4 kDa to about 6kDa poly(ethylene)glycol.

In some embodiments, the therapeutic nanoparticle is prepared by furtherfunctionalizing about 0.2 to about 30 weight percent poly(lactic)acid-poly(ethylene)glycol copolymer with a targeting ligand. In otherembodiments, the therapeutic nanoparticle is prepared by furtherfunctionalizing about 0.2 to about 30 weight percent poly(lactic)acid-co-poly(glycolic) acid-poly(ethylene)glycol copolymer with atargeting ligand. In certain embodiments, the targeting ligand iscovalently bound to the poly(ethylene)glycol.

In certain embodiments, the hydrophobic acid used in preparing thetherapeutic nanoparticle is a polyelectrolyte. For example, in someembodiments, the polyelectrolyte includes, but is not limited to, apoly(styrene sulfonic acid), polyacrylic acid, polymethacrylic acid, orcombinations thereof.

In certain embodiments, the therapeutic nanoparticle is prepared using amixture of two or more substantially hydrophobic acids. For example, insome embodiments, a mixture of two substantially hydrophobic acids, amixture of three substantially hydrophobic acids, a mixture of foursubstantially hydrophobic acids, or a mixture of five substantiallyhydrophobic acids may be used to prepare a therapeutic nanoparticle. Insome embodiments, the mixture of substantially hydrophobic acidscomprises oleic acid and cholic acid. In other embodiments, the mixtureof two substantially hydrophobic acids are oleic acid and cholic acid.

In certain embodiments, the therapeutic nanoparticle comprises thepolymer PLA-PEG and the mole ratio of PLA-PEG is about 5:1.

In some embodiments, a therapeutic nanoparticle is prepared by theprocess combining a first organic phase with a first aqueous solution toform a second phase; emulsifying the second phase to form an emulsionphase, wherein the emulsion phase comprises a first polymer, therapeuticagent, and a substantially hydrophobic acid; quenching of the emulsionphase thereby forming a quenched phase; and filtering the quenched phaseto recover the therapeutic nanoparticles, wherein the therapeutic agentis1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,the first organic phase comprises the therapeutic agent and pamoic acidin a weight ratio of therapeutic agent to pamoic acid of about 11:1 andPLA-PEG (in a 16:5 molar ratio) in a weight ratio of therapeutic agentto PLA-PEG of about 1:3 in an organic solvent comprising of benzylalcohol and ethyl acetate in a weight ratio of benzyl alcohol to ethylacetate of about 1.25 and the first aqueous solution comprises apolyoxyethylene (100) stearyl ether dissolved in benzyl alcohol in aweight ratio of 0.005:1 and combining the first organic phase and thefirst aqueous phase in a weight ratio of about 1:5 to form a secondphase and emulsifying the second phase formed therefrom and quenchingthe emulsion phase with 0.1 M citric acid in water solution at pH 4.5and concentrating the resulting product.

In other embodiments, the therapeutic nanoparticle comprises theaforementioned therapeutic agent or a pharmaceutically acceptable saltthereof and a polymer selected from diblock poly(lactic)acid-poly(ethylene)glycol copolymer or a diblock poly(lacticacid-co-glycolic acid)-poly(ethylene)glycol copolymer and combinationthereof.

In certain embodiments, the therapeutic nanoparticle has a targetingligand additionally present and the ligand is PLA-PEG-GL, wherein GL hasthe following structure:

In some embodiments, the therapeutic nanoparticle further comprises asolubilizer. In certain embodiments, the solubilizer is polysorbate 80.In other embodiments, the solubilizer is polyoxyethylene (100) stearylether.

In certain embodiments, the therapeutic nanoparticle comprises thetherapeutic agent1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,pamoic acid in a weight ratio of therapeutic agent to pamoic acid ofabout 1.8:1, PLA-PEG (in a 16:5 molar ratio) in a weight ratio oftherapeutic agent to PLA-PEG of about 1:3, and PLA-PEG-GL in a weightratio of PLA-PEG to PLA-PEG-GL of about 44:1. In other embodiments, thetherapeutic nanoparticle additionally comprises a solubilizer. Incertain such embodiments, the solubilizer is polyoxyethylene (100)stearyl ether.

In certain embodiments, the therapeutic nanoparticle comprises thetherapeutic agent1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,oleic acid in a weight ratio of therapeutic agent to oleic acid of about6:1, PLA-PEG (in a 16:5 molar ratio) in a weight ratio of therapeuticagent to PLA-PEG of about 1:7, and PLA-PEG-GL in a weight ratio ofPLA-PEG to PLA-PEG-GL of about 46:1. In some embodiments, thetherapeutic nanoparticle additionally comprises cholic acid. In otherembodiments, the therapeutic nanoparticle additionally comprises asolubilizer. In certain such embodiments, the solubilizer is polysorbate80.

In yet another aspect, a pharmaceutical composition comprising atherapeutic nanoparticle described herein and a pharmaceuticallyacceptable excipient is provided. The pharmaceutical composition maycomprise a plurality of contemplated therapeutic nanoparticles.

In certain embodiments, the pharmaceutical composition further comprisesa saccharide. For example, in some embodiments, the saccharide is adisaccharide selected from the group consisting of sucrose or trehalose,or a mixture thereof.

In certain embodiments, the pharmaceutical composition further comprisesa cyclodextrin. For example, in some embodiments, the cyclodextrinincludes, but is not limited to, α-cyclodextrin, β-cyclodextrin,γ-cyclodextrin, heptakis-(2,3,6-tri-O-benzyl)-β-cyclodextrin,heptakis-(2,3,6-tri-O-benzoyl)-β-cyclodextrin, or mixtures thereof.

In another aspect, a method of treating cancer in a subject in needthereof is provided. The method comprises administering to the subject atherapeutically effective amount of a pharmaceutical composition asdescribed herein. In some embodiments, the cancer is chronic myelogenousleukemia. In certain embodiments, the cancer includes, but is notlimited to chronic myelomonocytic leukemia, hypereosinophilic syndrome,renal cell carcinoma, hepatocellular carcinoma, Philadelphia chromosomepositive acute lymphoblastic leukemia, non-small cell lung cancer,pancreatic cancer, breast cancer, a solid tumor, mantle cell lymphoma,gastrointestinal stromal tumor, or head and neck cancer. In someembodiments, the cancer is breast cancer.

In still another aspect, a method of treating a gastrointestinal stromaltumor in a subject in need thereof is provided by administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition described herein.

In yet another aspect, a process for preparing a therapeuticnanoparticle is provided. The process comprises combining a firstorganic phase with a first aqueous solution to form a second phase;emulsifying the second phase to form an emulsion phase, wherein theemulsion phase comprises a first polymer, the aforementioned therapeuticagent, and a substantially hydrophobic acid; followed by quenching ofthe emulsion phase, thereby forming a quenched phase, and finallyfiltering the quenched phase to recover the therapeutic nanoparticles.

In some embodiments, the process further comprises combining theaforementioned therapeutic agent and the substantially hydrophobic acidin the second phase prior to emulsifying the second phase. In certainembodiments, the aforementioned therapeutic agent and the substantiallyhydrophobic acid form a hydrophobic ion pair prior to emulsifying thesecond phase. In certain other embodiments, the aforementionedtherapeutic agent and the substantially hydrophobic acid form ahydrophobic ion pair during emulsification of the second phase. Incertain embodiments, the process further comprises combining theaforementioned therapeutic agent and the substantially hydrophobic acidin the second phase substantially concurrently with emulsifying thesecond phase. For example, in some embodiments, the first organic phasecomprises the aforementioned therapeutic agent and the first aqueoussolution comprises the substantially hydrophobic acid.

In some embodiments, the aforementioned therapeutic agent, whenprotonated, has a first pK_(a), the substantially hydrophobic acid has asecond pK_(a), and the emulsion phase is quenched with an aqueoussolution having a pH equal to a pK_(a) unit between the first pK_(a) andthe second pK_(a). For example, in certain embodiments, the quenchedphase has a pH equal to a pK_(a) unit between the first pK_(a) and thesecond pK_(a). In other embodiments, the aforementioned therapeuticagent, when protonated, has a first pK_(a), the substantiallyhydrophobic acid has a second pK_(a), and the first aqueous solution hasa pH equal to a pK_(a) unit between the first pK_(a), and the secondpK_(a). In certain other embodiments, the pH is equal to a pK_(a) unitthat is about equidistant between the first pK_(a) and the secondpK_(a).

In some embodiments, the aforementioned therapeutic agent, whenprotonated, has a first pK_(a), the substantially hydrophobic acid has asecond pK_(a), and the emulsion phase is quenched with an aqueoussolution having a pH equal to a pK_(a) unit between the first pK_(a) andthe second pK_(a). For example, in certain embodiments, the quenchedphase has a pH equal to a pK_(a) unit between the first pK_(a) and thesecond pK_(a). In other embodiments, the aforementioned therapeuticagent, when protonated, has a first pK_(a), the substantiallyhydrophobic acid has a second pK_(a), and the first aqueous solution hasa pH equal to a pK_(a) unit between the first pK_(a), and the secondpK_(a). In certain other embodiments, the pH is equal to a pK_(a) unitthat is equidistant between the first pK_(a) and the second pK_(a).

In another aspect, there is provided a therapeutic nanoparticle asdescribed herein for use as a medicament in a subject.

In yet another aspect, there is provided a therapeutic nanoparticle asdescribed herein for use in the production of an anti-proliferativeeffect in a subject.

In still another aspect, there is provided a therapeutic nanoparticle asdescribed herein for use in a subject as an anti-invasive agent in thecontainment and/or treatment of solid tumor disease.

In yet another aspect, there is provided the use of a therapeuticnanoparticle as described herein in the prevention or treatment ofcancer in a subject.

In still another aspect, there is provided a therapeutic nanoparticle asdescribed herein for use in the prevention or treatment of cancer in asubject.

In yet another aspect, there is provided the use of a therapeuticnanoparticle as described herein in the manufacture of a medicament forthe prevention or treatment of cancer in a subject.

In still another aspect, there is provided the use of a therapeuticnanoparticle as described herein for the production of ananti-proliferative effect in a subject.

In yet another aspect, there is provided the use of a therapeuticnanoparticle as described herein in the manufacture of a medicament foruse in the production of an anti-proliferative effect in a subject.

In still another aspect, there is provided the use of a therapeuticnanoparticle as described herein in the manufacture of a medicament foruse in a subject as an anti-invasive agent in the containment and/ortreatment of solid tumor disease.

In yet another aspect, there is provided a method for producing ananti-proliferative effect in a subject in need of such treatment whichcomprises administering to said subject an effective amount of atherapeutic nanoparticle as described herein.

In still another aspect, there is provided a method for producing ananti-invasive effect by the containment and/or treatment of solid tumordisease in a subject in need of such treatment which comprisesadministering to said subject an effective amount of a therapeuticnanoparticle as described herein.

In yet another aspect, there is provided a therapeutic nanoparticle asdescribed herein for use in the prevention or treatment of solid tumordisease in a subject.

In still another aspect, there is provided the use of a therapeuticnanoparticle as described herein in the manufacture of a medicament foruse in the prevention or treatment of solid tumor disease in a subject.

In yet another aspect, there is provided a method for the prevention ortreatment of solid tumor disease in a subject in need of such treatmentwhich comprises administering to said subject an effective amount of atherapeutic nanoparticle as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is flow chart for an emulsion process for forming a disclosednanoparticle.

FIGS. 2A and 2B show flow diagrams for a disclosed emulsion process.

FIG. 3 depicts in vitro release profiles of nanoparticles of threeformulations described herein below identified as Formulations A, B, andC, respectively, each comprising1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea.Error bars indicate the standard deviation. Water bath temperature=37°C.

FIG. 4 depicts the pharmacokinetics of the nanoparticles of threeformulations described herein and identified as Formulations A, B, andC, respectively, each comprising1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,in Wistar Han Rats; (a) shows the pharmacokinetics of the nanoparticlerelative to free therapeutic agent, while (b) shows the same data withfree therapeutic agent omitted.

FIG. 5 depicts in vitro release profiles of Formulation A of the drug1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea.Error bars indicate the standard deviation. Water bath temperature=37°C.

FIG. 6 depicts in vitro release profiles of Formulation C of the drug1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureausing pH 4.5 citric acid buffered quench.

FIG. 7 depicts a MDAMB361 xenograft scheduling study in female SCID/bgmice dosed with Formulation B nanoparticles or1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea(naked API) once every 8 days versus once every 4 days.

FIGS. 8A, 8B, and 8C depict a MDAMB361 xenograft scheduling study infemale SCID/bg mice dosed with Formulation A, B, or C nanoparticles ornaked API once every 8 days versus once every 4 days, and in vivo pS6modulation studies with Formulation A, B, or C nanoparticles or1-{4-[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea(naked API). TA=therapeutic agent.

FIG. 9 depicts a WM266-4 model tumor growth inhibition study with femalenu/nu mice treated with Formulation B or C nanoparticles or1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea(naked API). TA=therapeutic agent.

FIG. 10 depicts an analysis of glucose and insulin levels in mice orrats after treatment with Formulation B or C nanoparticles or1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea(naked API).

DETAILED DESCRIPTION Definitions

The definitions set forth in this application are intended to clarifyterms used throughout this application.

The term “herein” means the entire application.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood by one of ordinaryskill in the art to which these inventions belong. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the inventions, suitable methods andmaterials are described below. The materials, methods and examples areillustrative only, and are not intended to be limiting. Allpublications, patents and other documents mentioned herein areincorporated by reference in their entirety.

Each embodiment of the inventions described herein may be taken alone orin combination with any one or more other embodiments of the inventions.

Throughout this application, the word “a” or “an” will be understood toimply the inclusion of one or more of the integers modified by thearticle “a” or “an.”

Throughout this application, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or groups of integers but not the exclusion of anyother integer or group of integers.

Throughout the application, where compositions are described as having,including, or comprising, specific components, it is contemplated thatcompositions also may consist essentially of, or consist of, the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso may consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the compositionsand methods described herein remains operable. Moreover, two or moresteps or actions can be conducted simultaneously.

The term “or” as used herein should be understood to mean “and/or”,unless the context clearly indicates otherwise.

The term “alkoxy” refers to an alkyl group, preferably a lower alkylgroup, having an oxygen attached thereto. Representative alkoxy groupsinclude methoxy, ethoxy, propoxy, tert-butoxy and the like.

Moreover, the term “alkyl” (or “lower alkyl”) as used herein is intendedto include both “unsubstituted alkyls” and “substituted alkyls”, thelatter of which refers to alkyl moieties having substituents replacing ahydrogen on one or more carbons of the hydrocarbon backbone. Suchsubstituents, if not otherwise specified, can include, for example, ahalogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl,a formyl, or an acyl), a thiocarbonyl (such as a thioester, athioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, aphosphonate, a phosphinate, an amino, an amido, an amidine, an imine, acyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, asulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, anaralkyl, or an aromatic moiety. It will be understood by those skilledin the art that the moieties substituted on the hydrocarbon chain canthemselves be substituted, if appropriate. For instance, thesubstituents of a substituted alkyl may include substituted andunsubstituted forms of amino, azido, imino, amido, phosphoryl (includingphosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido,sulfamoyl and sulfonate), and silyl groups, as well as ethers,alkylthiols, carbonyls (including ketones, aldehydes, carboxylates, andesters), —CF₃, —CN and the like.

The term “C_(x-y)” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups that contain from x to y carbons in the chain. Forexample, the term “C_(x-y)alkyl” refers to substituted or unsubstitutedsaturated hydrocarbon groups, including straight-chain alkyl andbranched-chain alkyl groups that contain from x to y carbons in thechain, including haloalkyl groups such as trifluoromethyl and2,2,2-trifluoroethyl, etc. C₀ alkyl indicates a hydrogen where the groupis in a terminal position, a bond if internal.

The term “amide”, as used herein, refers to a group

wherein each R³⁰ independently represent a hydrogen or hydrocarbylgroup, or two R³⁰ are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 12 atoms in the ringstructure.

The term “aryl” as used herein includes substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 5- to 7-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons orheteroatoms are common to two adjoining rings wherein at least one ofthe rings is aromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.

The terms “arylalkyl” or “aralkyl”, as used herein, refers to an alkylgroup substituted with an aryl group.

The term “azido” is art-recognized and refers to the group —N═N⁺═N⁻.

The term “carboxy”, as used herein, refers to a group represented by theformula —CO₂H.

The terms “halo” and “halogen” as used herein means halogen and includeschloro, fluoro, bromo, and iodo.

The term “hydrocarbyl”, as used herein, refers to a group that is bondedthrough a carbon atom that does not have a ═O or ═S substituent, andtypically has at least one carbon-hydrogen bond and a primarily carbonbackbone, but may optionally include heteroatoms. Thus, groups likemethyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to behydrocarbyl for the purposes of this application, but substituents suchas acetyl (which has a ═O substituent on the linking carbon) and ethoxy(which is linked through oxygen, not carbon) are not. Hydrocarbyl groupsinclude, but are not limited to aryl, carbocycle, heterocyclyl, alkyl,alkenyl, alkynyl, and combinations thereof.

The term “hydroxy”, as used herein, refers to an —OH group.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this application, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms.

Substituents can include any substituents described herein, for example,but not limited to, a halogen, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, anamido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl,an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic moiety. It will beunderstood by those skilled in the art that substituents can themselvesbe substituted, if appropriate. Unless specifically stated as“unsubstituted.” references to chemical moieties herein are understoodto include substituted variants. For example, reference to an “aryl”group or moiety implicitly includes both substituted and unsubstitutedvariants.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the application includesinstances where the circumstance occurs and instances where it does not.For example, the phrase “optionally substituted” means that anon-hydrogen substituent may or may not be present on a given atom, and,thus, the application includes structures wherein a non-hydrogensubstituent is present and structures wherein a non-hydrogen substituentis not present.

The terms “healthy” and “normal” are used interchangeably herein torefer to a subject or particular cell or tissue that is devoid (at leastto the limit of detection) of a disease condition.

Unless indicated to the contrary, the term “basic therapeutic agent” or“therapeutic agent” refers to the therapeutic agent1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl)}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor a pharmaceutically acceptable salt thereof. It has the structureshown below:

It is described in U.S. Pat. No. 8,039,469, the contents of which areincorporated by reference. The calculated log of the octanol:salinepartition coefficient (clog P)=1.24 (calculated partition coefficient).The log D, the distribution constant, at pH 6.5=0.212, while log D at pH7.4=1.08. As indicated above, it is a base. It is a protonatablenitrogen-containing therapeutic agent. As used herein, a “protonatablenitrogen-containing therapeutic agent” includes any pharmaceuticallyactive agent that contains at least one nitrogen-containing functionalgroup that is capable of being protonated. In other words, thetherapeutic agent has a nitrogen atom thereon which has a lone pair ofelectrons that could potentially accept a proton. The pK_(a) refers tothe acid dissociation constant on a logarithmic scale of thecorresponding protonated form of the therapeutic agent. In other words,if a proton (H+) were present at the nitrogen atoms where there is anarrow indicated, the therapeutic agent would have the pK_(a) indicatedbelow:

The pK_(a) information for the most basic nitrogen (bottom) and a lessbasic but still protonatable nitrogen (upper) are shown above. ACD is acalculated number using standard techniques known in the art, such asdescribed in Liao C Z, Nicklaus M C. Comparison of nine programspredicting pK(a) values of pharmaceutical substances. J. Chem. Inform.Model. 49(12):2801-2812, 2009. It has to be understood that the pK_(a)of the therapeutic agent refers to the protonated form thereof.

The therapeutic agent of the present invention possesses one or morechiral centers and the present invention includes each separateenantiomer of such compounds as well as mixtures of enantiomers. Wheremultiple chiral centers exist, the invention includes each combinationas well as mixtures thereof. All chiral, diastereomeric, and racemicforms of a structure are intended, unless the specific stereochemistryor isomeric form is specifically indicated. It is well known in the arthow to prepare optically active forms such as by resolution of racemicforms or by synthesis from optically active starting materials.

The term “substantially” when used in reference to a compound such as“hydrophobic acid” refers to the compound being present in at least 1%by weight or refers to a hydrophobic acid with a log P above 2. Ahydrophobic acid with a log P above 2 has a greater tendency topartition into the organic phase.

The term “hydrophobic acid” refers to a lipophilic acid which has a logof a −7 or greater, i.e., −6, −5, −4, −3, −2, −1, 0, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16.

The nanoparticles described herein may also contain pharmaceuticallyacceptable salts of the therapeutic agent. Representative“pharmaceutically acceptable salts” include but are not limited to,e.g., water-soluble and water-insoluble salts, such as the acetate,aluminum, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzathine(N,N′-dibenzylethylenediamine), benzenesulfonate, benzoate, bicarbonate,bismuth, bisulfate, bitartrate, borate, bromide, butyrate, calcium,calcium edetate, camsylate (camphorsulfonate), carbonate, chloride,choline, citrate, clavulariate, diethanolamine, dihydrochloride,diphosphate, edetate, edisylate (camphorsulfonate), esylate(ethanesulfonate), ethylenediamine, fumarate, gluceptate(glucoheptonate), gluconate, glucuronate, glutamate,hexafluorophosphate, hexylresorcinate,hydrabamine(N,N′-bis(dehydroabietyl)ethylenediamine), hydrobromide,hydrochloride, hydroxynaphthoate, 1-hydroxy-2-naphthoate,3-hydroxy-2-naphthoate, iodide, isothionate (2-hydroxyethanesulfonate),lactate, lactobionate, laurate, lauryl sulfate, lithium, magnesium,malate, maleate, mandelate, meglumine(1-deoxy-1-(methylamino)-D-glucitol), mesylate, methyl bromide,methylnitrate, methylsulfate, mucate, napsylate, nitrate,N-methylglucamine ammonium salt, oleate, oxalate, palmitate, pamoate(4,4′-methylenebis-3-hydroxy-2-naphthoate, or embonate), pantothenate,phosphate, picrate, polygalacturonate, potassium, propionate,p-toluenesulfonate, salicylate, sodium, stearate, subacetate, succinate,sulfate, sulfosaliculate, suramate, tannate, tartrate, teoclate(8-chloro-3,7-dihydro-1,3-dimethyl-1H-purine-2,6-dione), triethiodide,tromethamine(2-amino-2-(hydroxymethyl)-1,3-propanediol), valerate, andzinc salts.

When used herein below, unless indicated to the contrary, % is % byweight.

Described herein are polymeric nanoparticles that include the basictherapeutic agent and methods of making and using such therapeuticnanoparticles.

In some embodiments, inclusion (i.e., doping) of a substantiallyhydrophobic acid (e.g., a fatty acid and/or a bile acid) in a disclosednanoparticle and/or included in a nanoparticle preparation process mayresult in nanoparticles that include improved drug loading. Furthermore,in certain embodiments, nanoparticles that include and/or are preparedin the presence of the hydrophobic acid may exhibit improved controlledrelease properties. For example, disclosed nanoparticles may more slowlyrelease the therapeutic agent as compared to nanoparticles prepared inthe absence of the hydrophobic acid.

Without wishing to be bound by any theory, it is believed that thedisclosed nanoparticle formulations that include a hydrophobic acid(e.g., fatty acid and/or bile acid) have significantly improvedformulation properties (e.g., drug loading and/or release profile)through formation of a hydrophobic ion-pair (HIP), between thetherapeutic agent on the lone pair of electrons on one or more of thenitrogen atoms on the therapeutic agent indicated above, for example, onthe amine moiety and an acid. As used herein, a HIP is a pair ofoppositely charged ions held together by Coulombic attraction. Alsowithout wishing to be bound by any theory, in some embodiments, HIP canbe used to increase the hydrophobicity of the therapeutic agentcontaining ionizable groups (e.g., amines). When the therapeutic agenthas increased hydrophobicity, it is beneficial for nanoparticleformulations, for it results in a HIP formation that may provide highersolubility of the therapeutic agent in organic solvents. HIP formation,as contemplated herein, can result in nanoparticles having for example,increased drug loading. Slower release of the therapeutic agent from thenanoparticles may also occur, for example in some embodiments, due to adecrease in the therapeutic agent's solubility in aqueous solution.Furthermore, complexing the therapeutic agent with large hydrophobiccounter ions may slow diffusion of the therapeutic agent within thepolymeric matrix. Advantageously, HIP formation occurs without the needfor covalent conjugation of the hydrophobic group to the therapeuticagent.

Without wishing to be bound by any theory, it is believed that thestrength of the HIP impacts the drug load and release rate of thecontemplated nanoparticles. For example, the strength of the HIP may beincreased by increasing the magnitude of the difference between thepK_(a) of the protonated form of the therapeutic agent and the pK_(a) ofthe hydrophobic acid, as discussed in more detail below. Also withoutwishing to be bound by any theory, it is believed that the conditionsfor ion pair formation impact the drug load and release rate of thecontemplated nanoparticles.

Nanoparticles disclosed herein include one, two, three or morebiocompatible and/or biodegradable polymers. For example, a contemplatednanoparticle may include about 35 to about 99.75 weight percent in someembodiments; about 50 to about 99.75 weight percent, in some otherembodiments; about 50 to about 99.5 weight percent, in some embodiments;about 50 to about 99 weight percent in still other embodiments; about 50to about 98 weight percent in further embodiments; about 50 to about 97weight percent in still further embodiments; about 50 to about 96 weightpercent in additional embodiments; about 50 to about 95 weight percentin other embodiments, about 50 to about 94 weight percent in still otherembodiments; about 50 to about 93 weight percent in other embodiments;about 50 to about 92 weight percent in still other embodiments; about 50to about 91 weight percent, in some embodiments about 50 to about 90weight percent; in some embodiments, about 50 to about 85 weightpercent; in some embodiments about 60 to about 85 weight percent; insome embodiments, about 65 to about 85 weight percent; and in someembodiments, about 50 to about 80 weight percent of one or more blockcopolymers that include a biodegradable polymer and poly(ethyleneglycol) (PEG), and about 0 to about 50 weight percent of a biodegradablehomopolymer.

In some embodiments, a contemplated nanoparticle may include 35 to 99.75weight percent in some embodiments; 50 to 99.75 weight percent, in someother embodiments; 50 to 99.5 weight percent, in some embodiments; 50 to99 weight percent in still other embodiments; 50 to 98 weight percent infurther embodiments; 50 to 97 weight percent in still furtherembodiments; 50 to 96 weight percent in additional embodiments; 50 to 95weight percent in other embodiments, 50 to 94 weight percent in stillother embodiments; 50 to 93 weight percent in other embodiments; 50 to92 weight percent in still other embodiments; 50 to 91 weight percent,in some embodiments 50 to 90 weight percent; in some embodiments, 50 to85 weight percent; in some embodiments 60 to 85 weight percent; in someembodiments, 65 to 85 weight percent; and in some embodiments, 50 to 80weight percent of one or more block copolymers that include abiodegradable polymer and poly(ethylene glycol) (PEG), and 0 to 50weight percent of a biodegradable homopolymer.

In some embodiments, disclosed nanoparticles may include about 0.2 toabout 35 weight percent, about 0.2 to about 25 weight percent, about 0.2to about 20 weight percent, about 0.2 to about 10 weight percent, about0.2 to about 5 weight percent, about 0.5 to about 5 weight percent,about 0.75 to about 5 weight percent, about 1 to about 5 weight percent,about 2 to about 5 weight percent, about 3 to about 5 weight percent,about 1 to about 20 weight percent, about 2 to about 20 weight percent,about 3 to about 20 weight percent, about 4 to about 20 weight percent,about 5 to about 20 weight percent, about 1 to about 15 weight percent,about 2 to about 15 weight percent, about 3 to about 15 weight percent,about 4 to about 15 weight percent, about 5 to about 15 weight percent,about 1 to about 10 weight percent, about 2 to about 10 weight percent,about 3 to about 10 weight percent, about 4 to about 10 weight percent,about 5 to about 10 weight percent, about 10 to about 30 weight percent,or about 15 to about 25 weight percent of the therapeutic agent. In someembodiments the disclosed nanoparticles include about 0.2, about 0.3,about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about9, about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23,about 24, about 25, about 26, about 27, about 28, about 29, or about 30weight percent of the therapeutic agent.

In certain embodiments, disclosed nanoparticles may include 0.2 to 35weight percent, 0.2 to 25 weight percent, 0.2 to 20 weight percent, 0.2to 10 weight percent, 0.2 to 5 weight percent, 0.5 to 5 weight percent,0.75 to 5 weight percent, 1 to 5 weight percent, 2 to 5 weight percent,3 to 5 weight percent, 1 to 20 weight percent, 2 to 20 weight percent, 3to 20 weight percent, 4 to 20 weight percent, 5 to 20 weight percent, 1to 15 weight percent, 2 to 15 weight percent, 3 to 15 weight percent, 4to 15 weight percent, 5 to 15 weight percent, 1 to 10 weight percent, 2to 10 weight percent, 3 to 10 weight percent, 4 to 10 weight percent, 5to 10 weight percent, 10 to 30 weight percent, or 15 to 25 weightpercent of the therapeutic agent. In some embodiments the disclosednanoparticles include 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 weight percent of the therapeuticagent.

In certain embodiments, disclosed nanoparticles comprise a hydrophobicacid (e.g., a fatty acid and/or bile acid) and/or are prepared by aprocess that includes a hydrophobic acid. Such nanoparticles may have ahigher drug loading than nanoparticles prepared by a process without ahydrophobic acid. For example, drug loading (e.g., by weight) ofdisclosed nanoparticles prepared by a process comprising the hydrophobicacid may be between about 2 times to about 10 times higher, or evenmore, than disclosed nanoparticles prepared by a process without thehydrophobic acid. In some embodiments, the drug loading (by weight) ofdisclosed nanoparticles prepared by a first process comprising thehydrophobic acid may be at least about 2 times higher, at least about 3times higher, at least about 4 times higher, at least about 5 timeshigher, or at least about 10 times higher than disclosed nanoparticlesprepared by a second process, where the second process is identical tothe first process except that the second process does not include thehydrophobic acid.

In certain embodiments, drug loading (e.g., by weight) of disclosednanoparticles prepared by a process comprising the hydrophobic acid maybe between 2 times to 10 times higher, or even more, than disclosednanoparticles prepared by a process without the hydrophobic acid. Insome embodiments, the drug loading (by weight) of disclosednanoparticles prepared by a first process comprising the hydrophobicacid may be at least 2 times higher, at least 3 times higher, at least 4times higher, at least 5 times higher, or at least 10 times higher thandisclosed nanoparticles prepared by a second process, where the secondprocess is identical to the first process except that the second processdoes not include the hydrophobic acid.

Any suitable hydrophobic acid is contemplated. In some embodiments, thehydrophobic acid may be a carboxylic acid (e.g., a monocarboxylic acid,dicarboxylic acid, tricarboxylic acid, or the like), a sulfinic acid, asulfenic acid, or a sulfonic acid. In some cases, a contemplatedhydrophobic acid may include a mixture of two or more acids. Forexample, in certain embodiments, the hydrophobic acid may comprise amixture of two substantially hydrophobic acids, in some embodiments amixture of three substantially hydrophobic acids, in some embodiments amixture of four substantially hydrophobic acids, or in some embodimentsfive substantially hydrophobic acids. In some embodiments, the mixtureof substantially hydrophobic acids comprises oleic acid and cholic acid.In other embodiments, the mixture of two hydrophobic acids is oleic acidand cholic acid.

In some cases, a salt of a hydrophobic acid may be used in aformulation.

For example, a disclosed carboxylic acid may be an aliphatic carboxylicacid (e.g., a carboxylic acid having a cyclic or acyclic, branched orunbranched, hydrocarbon chain). Disclosed carboxylic acids may, in someembodiments, be substituted with one or more functional groupsincluding, but not limited to, halogen (i.e., F, Cl, Br, and I),sulfonyl, nitro, and oxo. In certain embodiments, a disclosed carboxylicacid may be unsubstituted.

Exemplary carboxylic acids may include a substituted or unsubstitutedfatty acid (e.g., C₆-C₅₀ fatty acid). In some instances, the fatty acidmay be a C₁₀-C₂₀ fatty acid. In other instances, the fatty acid may be aC₁₅-C₂₀ fatty acid. The fatty acid may, in some cases, be saturated. Inother embodiments, the fatty acid may be unsaturated. For instance, thefatty acid may be a monounsaturated fatty acid or a polyunsaturatedfatty acid. In some embodiments, a double bond of an unsaturated fattyacid group can be in the cis conformation. In some embodiments, a doublebond of an unsaturated fatty acid can be in the trans conformation.Unsaturated fatty acids include, but are not limited to, omega-3,omega-6, or omega-9 fatty acids.

Non-limiting examples of saturated fatty acids include caproic acid,enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoicacid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid,palmitic acid, margaric acid, stearic acid, nonadecanoic acid, arachidicacid, heneicosanoic acid, behenic acid, tricosanoic acid, lignocericacid, pentacosanoic acid, cerotic acid, heptacosanoic acid, montanicacid, nonacosanoic acid, melissic acid, henatriacontanoic acid,lacceroic acid, psyllic acid, geddic acid, ceroplastic acid,hexatriacontanoic acid, or combinations thereof.

Non-limiting examples of unsaturated fatty acids includehexadecatrienoic acid, alpha-linolenic acid, stearidonic acid,eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid,heneicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid,tetracosapentaenoic acid, tetracosahexaenoic acid, linoleic acid,gamma-linolenic acid, eicosadienoic acid, dihomo-gamma-linolenic acid,arachidonic acid, docosadienoic acid, adrenic acid, docosapentaenoicacid, tetracosatetraenoic acid, tetracosapentaenoic acid, oleic acid(pK_(a)=˜4-5; log P=6.78), eicosenoic acid, mead acid, erucic acid,nervonic acid, rumenic acid, α-calendic acid, β-calendic acid, jacaricacid, α-eleostearic acid, β-eleostearic acid, catalpic acid, punicicacid, rumelenic acid, α-parinaric acid, β-parinaric acid,bosseopentaenoic acid, pinolenic acid, podocarpic acid, palmitoleicacid, vaccenic acid, gadoleic acid, erucic acid, or combinationsthereof.

Other non-limiting examples of hydrophobic acids include aromatic acids,such as 1-hydroxy-2-naphthoic acid (i.e., xinafoic acid) (pK_(a)=˜2-3;log P=2.97), naphthalene-1,5-disulfonic acid (pK_(a)=−2; log P=1.3),naphthalene-2-sulfonic acid (pK_(a)=−1.8; log P=2.1), pamoic acid(pK_(a)=2.4; log P=6.17), cinnamic acid, phenylacetic acid,(±)-camphor-10-sulfonic acid, dodecylbenzenesulfonic acid (pK_(a),=−1.8; log P=6.6), or combinations thereof. Other non-limiting examplesof hydrophobic acids include dodecylsulfuric acid (pK_(a)=−0.09; logP=4.5), dioctyl sulfosuccinic acid (i.e., docusate acid) (pK_(a)=−0.8;log P=5.2), dioleoyl phosphatidic acid (pK_(a)=˜2), or VitaminD₃-sulfate (pK_(a)=−1.5).

In some embodiments, the hydrophobic acid may be a bile acid.Non-limiting examples of bile acids include chenodeoxycholic acid,ursodeoxycholic acid, deoxycholic acid (pK_(a)=4.65; log P=3.79),hycholic acid, beta-muricholic acid, cholic acid (pK_(a), =˜4.5; logP=2.48), taurocholic acid, cholesteryl sulfate (pK_(a), =−1.4),lithocholic acid, an amino acid-conjugated bile acid, or combinationsthereof. An amino-acid conjugated bile acid may be conjugated to anysuitable amino acid. In some embodiments, the amino acid-conjugated bileacid is a glycine-conjugated bile acid or a taurine-conjugated bileacid.

In certain instances, the hydrophobic acid may be a polyelectrolyte. Forexample, the polyelectrolyte may be a polysulfonic acid (e.g.,poly(styrene sulfonic acid) or dextran sulfate) or a polycarboxylic acid(e.g., polyacrylic acid or polymethacrylic acid).

In some instances, a contemplated hydrophobic acid may have a molecularweight of less than about 1000 Da, in some embodiments less than about500 Da, in some embodiments less than about 400 Da, in some embodimentsless than about 300 Da, in some embodiments less than about 250 Da, insome embodiments less than about 200 Da, and in some embodiments lessthan about 150 Da. In some cases, the hydrophobic acid may have amolecular weight of between about 100 Da and about 1000 Da, in someembodiments between about 200 Da and about 800 Da, in some embodimentsbetween about 200 Da and about 600 Da, in some embodiments between about100 Da and about 300 Da, in some embodiments between about 200 Da andabout 400 Da, in some embodiments between about 300 Da and about 500 Da,and in some embodiments between about 300 Da and about 1000 Da. Incertain embodiments, a contemplated acid may have a molecular weight ofgreater than about 200 kDa, in some embodiments greater than about 300Da, in some embodiments greater than about 400 Da, and in someembodiments greater than about 500 Da. In certain embodiments, therelease rate of a therapeutic agent from a nanoparticle can be slowed byincreasing the molecular weight of the hydrophobic acid used in thenanoparticle formulation.

In some instances, a contemplated hydrophobic acid may have a molecularweight of less than 1000 Da, in some embodiments less than 500 Da, insome embodiments less than 400 Da, in some embodiments less than 300 Da,in some embodiments less than 250 Da, in some embodiments less than 200Da, and in some embodiments less than 150 Da. In some cases, thehydrophobic acid may have a molecular weight of between 100 Da and 1000Da, in some embodiments between 200 Da and 800 Da, in some embodimentsbetween 200 Da and 600 Da, in some embodiments between 100 Da and 300Da, in some embodiments between 200 Da and 400 Da, in some embodimentsbetween 300 Da and 500 Da, and in some embodiments between 300 Da and1000 Da. In certain embodiments, a contemplated acid may have amolecular weight of greater than 200 kDa, in some embodiments greaterthan 300 Da, in some embodiments greater than 400 Da, and in someembodiments greater than 500 Da.

In some embodiments, a hydrophobic acid may be chosen, at least in part,on the basis of the strength of the acid. For example, the hydrophobicacid may have an acid dissociation constant in water (pK_(a)) of about−5 to about 7, in some embodiments about −3 to about 5, in someembodiments about −3 to about 4, in some embodiments about −3 to about3.5, in some embodiments about −3 to about 3, in some embodiments about−3 to about 2, in some embodiments about −3 to about 1, in someembodiments about −3 to about 0.5, in some embodiments, about −1.0 toabout 5.0, in some embodiments about −0.5 to about 0.5, in someembodiments about 1 to about 7, in some embodiments about 2 to about 7,in some embodiments about 2.0 to about 5.0, in some embodiments about 3to about 7, in some embodiments about 4 to about 6, in some embodimentsabout 4 to about 5.5, in some embodiments about 4 to about 5, and insome embodiments about 4.5 to about 5, determined at 25° C. In someembodiments, the acid may have a pK_(a) of less than about 7, less thanabout 5, less than about 3.5, less than about 3, less than about 2, lessthan about 1, or less than about 0, determined at 25° C.

In some embodiments, the hydrophobic acid may have an acid dissociationconstant in water (pK_(a)) of −5 to 7, in some embodiments −3 to 5, insome embodiments −3 to 4, in some embodiments −3 to 3.5, in someembodiments −3 to 3, in some embodiments −3 to 2, in some embodiments −3to 1, in some embodiments −3 to 0.5, in some embodiments, −1.0 to 5.0,in some embodiments −0.5 to 0.5, in some embodiments 1 to 7, in someembodiments 2 to 7, in some embodiments 2.0 to 5.0, in some embodiments3 to 7, in some embodiments 4 to 6, in some embodiments 4 to 5.5, insome embodiments 4 to 5, and in some embodiments 4.5 to 5, determined at25° C. In some embodiments, the acid may have a pK_(a) of less than 7,less than 5, less than 3.5, less than 3, less than 2, less than 1, orless than 0, determined at 25° C.

In certain embodiments, the hydrophobic acid may be chosen, at least inpart, on the basis of the difference between the pK_(a) of thehydrophobic acid and the pK_(a) of the protonated therapeutic agent. Forexample, in some instances, the difference between the pK_(a) of thehydrophobic acid and the pK_(a) of the protonated therapeutic agent maybe between about 1 pK_(a) unit and about 15 pK_(a) units, in someembodiments between about 1 pK_(a) unit and about 10 pK_(a) units, insome embodiments between about 1 pK_(a) unit and about 5 pK_(a) units,in some embodiments between about 1 pK_(a) unit and about 3 pK_(a)units, in some embodiments between about 1 pK_(a) unit and about 2pK_(a) units, in some embodiments between about 2 pK_(a) units and about15 pK_(a) units, in some embodiments between about 2 pK_(a) units andabout 10 pK_(a) units, in some embodiments between about 2 pK_(a) unitsand about 5 pK_(a) units, in some embodiments between about 2 pK_(a)units and about 3 pK_(a) units, in some embodiments between about 3pK_(a) units and about 15 pK_(a) units, in some embodiments betweenabout 3 pK_(a) units and about 10 pK_(a) units, in some embodimentsbetween about 3 pK_(a) units and about 5 pK_(a) units, in someembodiments between about 4 pK_(a) units and about 15 pK_(a) units, insome embodiments between about 4 pK_(a) units and about 10 pK_(a) units,in some embodiments between about 4 pK_(a) units and about 6 pK_(a)units, in some embodiments between about 5 pK_(a) units and about 15pK_(a) units, in some embodiments between about 5 pK_(a) units and about10 pK_(a) units, in some embodiments between about 5 pK_(a) units andabout 7 pK_(a) units, in some embodiments between about 7 pK_(a) unitsand about 15 pK_(a) units, in some embodiments between about 7 pK_(a)units and about 9 pK_(a) units, in some embodiments between about 9pK_(a) units and about 15 pK_(a) units, in some embodiments betweenabout 9 pK_(a) units and about 11 pK_(a) units, in some embodimentsbetween about 11 pK_(a) units and about 13 pK_(a) units, and in someembodiments between about 13 pK_(a) units and about 15 pK_(a) units,determined at 25° C.

In certain embodiments, the difference between the pK_(a) of thehydrophobic acid and the pK_(a) of the protonated therapeutic agent maybe between 1 pK_(a) unit and 15 pK_(a) units, in some embodimentsbetween 1 pK_(a) unit and 10 pK_(a) units, in some embodiments between 1pK_(a) unit and 5 pK_(a) units, in some embodiments between 1 pK_(a)unit and 3 pK_(a) units, in some embodiments between 1 pK_(a) unit and 2pK_(a) units, in some embodiments between 2 pK_(a) units and 15 pK_(a)units, in some embodiments between 2 pK_(a) units and 10 pK_(a) units,in some embodiments between 2 pK_(a) units and 5 pK_(a) units, in someembodiments between 2 pK_(a), units and 3 pK_(a) units, in someembodiments between 3 pK_(a) units and 15 pK_(a) units, in someembodiments between 3 pK_(a) units and 10 pK_(a) units, in someembodiments between 3 pK_(a) units and 5 pK_(a) units, in someembodiments between 4 pK_(a) units and 15 pK_(a) units, in someembodiments between 4 pK_(a) units and 10 pK_(a) units, in someembodiments between 4 pK_(a) units and 6 pK_(a) units, in someembodiments between 5 pK_(a) units and 15 pK_(a) units, in someembodiments between 5 pK_(a) units and 10 pK_(a) units, in someembodiments between 5 pK_(a) units and 7 pK_(a) units, in someembodiments between 7 pK_(a) units and 15 pK_(a) units, in someembodiments between 7 pK_(a) units and 9 pK_(a) units, in someembodiments between 9 pK_(a) units and 15 pK_(a) units, in someembodiments between 9 pK_(a) units and 11 pK_(a) units, in someembodiments between 11 pK_(a) units and 13 pK_(a) units, and in someembodiments between 13 pK_(a) units and 15 pK_(a) units, determined at25° C.

In some instances, the difference between the pK_(a) of the hydrophobicacid and the pK_(a) of the protonated therapeutic agent may be at leastabout 1 pK_(a) unit, in some embodiments at least about 2 pK_(a) units,in some embodiments at least about 3 pK_(a) units, in some embodimentsat least about 4 pK_(a) units, in some embodiments at least about 5pK_(a) units, in some embodiments at least about 6 pK_(a) units, in someembodiments at least about 7 pK_(a) units, in some embodiments at leastabout 8 pK_(a) units, in some embodiments at least about 9 pK_(a) units,in some embodiments at least about 10 pK_(a) units, and in someembodiments at least about 15 pK_(a) units, determined at 25° C.

In some embodiments, the difference between the pK_(a) of thehydrophobic acid and the pK_(a) of the protonated therapeutic agent maybe at least 1 pK_(a) unit, in some embodiments at least 2 pK_(a) units,in some embodiments at least 3 pK_(a) units, in some embodiments atleast 4 pK_(a) units, in some embodiments at least 5 pK_(a) units, insome embodiments at least 6 pK_(a) units, in some embodiments at least 7pK_(a) units, in some embodiments at least 8 pK_(a) units, in someembodiments at least 9 pK_(a) units, in some embodiments at least 10pK_(a) units, and in some embodiments at least 15 pK_(a) units,determined at 25° C.

In some embodiments, the hydrophobic acid may have a log P of betweenabout 2 and about 15, in some embodiments between about 5 and about 15,in some embodiments between about 5 and about 10, in some embodimentsbetween about 2 and about 8, in some embodiments between about 4 andabout 8, in some embodiments between about 2 and about 7, or in someembodiments between about 4 and about 7. In some instances, thehydrophobic acid may have a log P greater than about 2, greater thanabout 4, greater than about 5, or greater than about 6.

In some embodiments, the hydrophobic acid may have a log P of between 2and 15, in some embodiments between 5 and 15, in some embodimentsbetween 5 and 10, in some embodiments between 2 and 8, in someembodiments between 4 and 8, in some embodiments between 2 and 7, or insome embodiments between 4 and 7. In some instances, the hydrophobicacid may have a log P greater than 2, greater than 4, greater than 5, orgreater than 6.

In some embodiments, a contemplated hydrophobic acid may have a phasetransition temperature that is advantageous, for example, for improvingthe properties of the therapeutic nanoparticles. For instance, thehydrophobic acid may have a melting point of less than about 350° C., insome cases less than about 300° C., in some cases less than about 100°C., and in some cases less than about 50° C. In certain embodiments, thehydrophobic acid may have a melting point of between about 5° C. andabout 25° C., in some cases between about 15° C. and about 50° C., insome cases between about 30° C. and about 100° C., in some cases betweenabout 75° C. and about 150° C., in some cases between about 125° C. andabout 200° C., in some cases between about 150° C. and about 250° C., insome cases between about 200° C. and about 300° C., and in some casesbetween about 250° C. and about 350° C. In some cases, the hydrophobicacid may have a melting point of less than about 15° C., in some casesless than about 10° C., or in some cases less than about 0° C. Incertain embodiments, the hydrophobic acid may have a melting point ofbetween about −30° C. and about 0° C. or in some cases between about−20° C. and about −10° C.

In some embodiments, the hydrophobic acid may have a melting point ofless than 350° C., in some cases less than 300° C., in some cases lessthan 100° C., and in some cases less than 50° C. In certain embodiments,the hydrophobic acid may have a melting point of between 5° C. and 25°C., in some cases between 15° C. and 50° C., in some cases between 30°C. and 100° C. in some cases between 75° C. and 150° C., in some casesbetween 125° C. and 200° C., in some cases between 150° C. and 250° C.,in some cases between 200° C. and 300° C., and in some cases between250° C. and 350° C. In some cases, the hydrophobic acid may have amelting point of less than 15° C., in some cases less than 10° C., or insome cases less than 0° C. In certain embodiments, the hydrophobic acidmay have a melting point of between −30° C. and 0° C. or in some casesbetween −20° C. and −10° C.

For example, hydrophobic acid for use in methods and nanoparticlesdisclosed herein may be chosen, at least in part, on the basis of thesolubility of the therapeutic agent in a solvent comprising the acid.For example, in some embodiments, depending on the solvent, thetherapeutic agent dissolved in a solvent comprising the acid may have asolubility of between about 15 mg/mL to about 200 mg/mL, between about20 mg/mL to about 200 mg/mL, between about 25 mg/mL to about 200 mg/mL,between about 50 mg/mL to about 200 mg/mL, between about 75 mg/mL toabout 200 mg/mL, between about 100 mg/mL to about 200 mg/mL, betweenabout 125 mg/mL to about 175 mg/mL, between about 15 mg/mL to about 50mg/mL, between about 25 mg/mL to about 75 mg/mL. In some embodiments,the therapeutic agent dissolved in a solvent containing the hydrophobicacid may have a solubility greater than about 10 mg/mL, greater thanabout 50 mg/mL, or greater than about 100 mg/mL. In some embodiments,the therapeutic agent dissolved in a solvent containing the hydrophobicacid (e.g., a first solution consisting of the therapeutic agent,solvent, and hydrophobic acid) may have a solubility of at least about 2times greater, in some embodiments at least about 5 times greater, insome embodiments at least about 10 times greater, in some embodiments atleast about 20 times greater, in some embodiments about 2 times to about20 times greater or in some embodiments about 10 times to about 20 timesgreater than when the therapeutic agent is dissolved in a solvent thatdoes not contain the hydrophobic acid (e.g., a second solutionconsisting of the therapeutic agent and the solvent).

In some embodiments, depending on the solvent, the therapeutic agentdissolved in a solvent comprising the acid may have a solubility ofbetween 15 mg/mL to 200 mg/mL, between 20 mg/mL to 200 mg/mL, between 25mg/mL to 200 mg/mL, between 50 mg/mL to 200 mg/mL, between 75 mg/mL to200 mg/mL, between 100 mg/mL to 200 mg/mL, between 125 mg/mL to 175mg/mL, between 15 mg/mL to 50 mg/mL, between 25 mg/mL to 75 mg/mL. Insome embodiments, the therapeutic agent dissolved in a solventcontaining the hydrophobic acid may have a solubility greater than 10mg/mL, greater than 50 mg/mL, or greater than 100 mg/mL. In someembodiments, the therapeutic agent dissolved in a solvent containing thehydrophobic acid (e.g., a first solution consisting of the therapeuticagent, solvent, and hydrophobic acid) may have a solubility of at least2 times greater, in some embodiments at least 5 times greater, in someembodiments at least 10 times greater, in some embodiments at least 20times greater, in some embodiments 2 times to 20 times greater or insome embodiments 10 times to 20 times greater than when the therapeuticagent is dissolved in a solvent that does not contain the hydrophobicacid (e.g., a second solution consisting of the therapeutic agent andthe solvent).

In some instances, the concentration of hydrophobic acid in a drugsolution (i.e., the therapeutic agent solution) may range from about 1weight percent to about 30 weight percent, in some embodiments, fromabout 2 weight percent to about 30 weight percent, in some embodiments,from about 3 weight percent to about 30 weight percent, in someembodiments, from about 4 weight percent to about 30 weight percent, insome embodiments, from about 5 weight percent to about 30 weightpercent, in some embodiments, from about 6 weight percent to about 30weight percent, in some embodiments, from about 8 weight percent toabout 30 weight percent, in some embodiments, from about 10 weightpercent to about 30 weight percent, in some embodiments, from about 12weight percent to about 30 weight percent, in some embodiments, fromabout 14 weight percent to about 30 weight percent, in some embodiments,from about 16 weight percent to about 30 weight percent, in someembodiments, from about 1 weight percent to about 5 weight percent, insome embodiments, from about 3 weight percent to about 9 weight percent,in some embodiments, from about 6 weight percent to about 12 weightpercent, in some embodiments, from about 9 weight percent to about 15weight percent, in some embodiments, from about 12 weight percent toabout 18 weight percent, and in some embodiments, from about 15 weightpercent to about 21 weight percent. In certain embodiments, theconcentration of hydrophobic acid in a drug solution may be about 1weight percent or greater, in some embodiments about 2 weight percent orgreater, in some embodiments about 3 weight percent or greater, in someembodiments about 5 weight percent or greater, in some embodiments about10 weight percent or greater, in some embodiments about 15 weightpercent or greater, and in some embodiments about 20 weight percent orgreater.

In some instances, the concentration of hydrophobic acid in a drugsolution (i.e., the therapeutic agent solution) may range from 1 weightpercent to 30 weight percent, in some embodiments, from 2 weight percentto 30 weight percent, in some embodiments, from 3 weight percent to 30weight percent, in some embodiments, from 4 weight percent to 30 weightpercent, in some embodiments, from 5 weight percent to 30 weightpercent, in some embodiments, from 6 weight percent to 30 weightpercent, in some embodiments, from 8 weight percent to 30 weightpercent, in some embodiments, from 10 weight percent to 30 weightpercent, in some embodiments, from 12 weight percent to 30 weightpercent, in some embodiments, from 14 weight percent to 30 weightpercent, in some embodiments, from 16 weight percent to 30 weightpercent, in some embodiments, from 1 weight percent to 5 weight percent,in some embodiments, from 3 weight percent to 9 weight percent, in someembodiments, from 6 weight percent to 12 weight percent, in someembodiments, from 9 weight percent to 15 weight percent, in someembodiments, from 12 weight percent to 18 weight percent, and in someembodiments, from 15 weight percent to 21 weight percent. In certainembodiments, the concentration of hydrophobic acid in a drug solutionmay be 1 weight percent or greater, in some embodiments 2 weight percentor greater, in some embodiments 3 weight percent or greater, in someembodiments 5 weight percent or greater, in some embodiments 10 weightpercent or greater, in some embodiments 15 weight percent or greater,and in some embodiments 20 weight percent or greater.

In certain embodiments, the molar ratio of hydrophobic acid totherapeutic agent (e.g., initially during formulation of thenanoparticles and/or in the nanoparticles) may range from about 0.25:1to about 6:1, in some embodiments from about 0.25:1 to about 5:1, insome embodiments from about 0.25:1 to about 4:1, in some embodiments,from about 0.25:1 to about 3:1, in some embodiments from about 0.25:1 toabout 2:1, in some embodiments, from about 0.25:1 to about 1.5:1, insome embodiments, from about 0.25:1 to about 1:1, in some embodiments,from about 0.25:1 to about 0.5:1, in some embodiments from about 0.5:1to about 6:1, in some embodiments, from about 0.5:1 to about 5:1, insome embodiments, from about 0.5:1 to about 4:1, in some embodimentsfrom about 0.5:1 to about 3:1, in some embodiments from about 0.5:1 toabout 2:1, in some embodiments from about 0.5:1 to about 1.5:1, in someembodiments from about 0.5:1 to about 1:1, in some embodiments, fromabout 0.5:1 to about 0.75:1, in some embodiments, from about 0.75:1 toabout 2:1, in some embodiments from about 0.75:1 to about 1.5:1, in someembodiments, from about 0.75:1 to about 1.25:1, in some embodiments,from about 0.9:1 to about 1.1:1, in some embodiments, from about 0.95:1to about 1.05:1, in some embodiments, about 1:1, in some embodimentsfrom about 0.75:1 to about 1:1, in some embodiments from about 1:1 toabout 6:1, in some embodiments, from about 1:1 to about 5:1, in someembodiments from about 1:1 to about 4:1, in some embodiments, from about1:1 to about 3:1, in some embodiments, from about 1:1 to about 2:1, insome embodiments from about 1:1 to about 1.5:1, in some embodiments,from about 1.5:1 to about 6:1, in some embodiments, from about 1.5:1 toabout 5:1, in some embodiments from about 1.5:1 to about 4:1, in someembodiments from about 1.5:1 to about 3:1, in some embodiments fromabout 2:1 to about 6:1, in some embodiments from about 2:1 to about 4:1,in some embodiments, from about 3:1 to about 6:1, in some embodiments,from about 3:1 to about 5:1, and in some embodiments, from about 4:1 toabout 6:1.

In certain embodiments, the molar ratio of hydrophobic acid totherapeutic agent (e.g., initially during formulation of thenanoparticles and/or in the nanoparticles) may range from 0.25:1 to 6:1,in some embodiments from 0.25:1 to 5:1, in some embodiments from 0.25:1to 4:1, in some embodiments, from 0.25:1 to 3:1, in some embodimentsfrom 0.25:1 to 2:1, in some embodiments, from 0.25:1 to 1.5:1, in someembodiments, from 0.25:1 to 1:1, in some embodiments, from 0.25:1 to0.5:1, in some embodiments from 0.5:1 to 6:1, in some embodiments, from0.5:1 to 5:1, in some embodiments, from 0.5:1 to 4:1, in someembodiments from 0.5:1 to 3:1, in some embodiments from 0.5:1 to 2:1, insome embodiments from 0.5:1 to 1.5:1, in some embodiments from 0.5:1 to1:1, in some embodiments, from 0.5:1 to 0.75:1, in some embodiments,from 0.75:1 to 2:1, in some embodiments from 0.75:1 to 1.5:1, in someembodiments, from 0.75:1 to 1.25:1, in some embodiments, from 0.9:1 to1.1:1, in some embodiments, from 0.95:1 to 1.05:1, in some embodiments,1:1, in some embodiments from 0.75:1 to 1:1, in some embodiments from1:1 to 6:1, in some embodiments, from 1:1 to 5:1, in some embodimentsfrom 1:1 to 4:1, in some embodiments, from 1:1 to 3:1, in someembodiments, from 1:1 to 2:1, in some embodiments from 1:1 to 1.5:1, insome embodiments, from 1.5:1 to 6:1, in some embodiments, from 1.5:1 to5:1, in some embodiments from 1.5:1 to 4:1, in some embodiments from1.5:1 to 3:1, in some embodiments from 2:1 to 6:1, in some embodimentsfrom 2:1 to 4:1, in some embodiments, from 3:1 to 6:1, in someembodiments, from 3:1 to 5:1, and in some embodiments, from 4:1 to 6:1.

In some instances, the initial molar ratio of hydrophobic acid totherapeutic agent (i.e., during formulation of the nanoparticles) may bedifferent from the molar ratio of hydrophobic acid to therapeutic agentin the nanoparticles (i.e., after removal of unencapsulated hydrophobicacid and therapeutic agent). In other instances, the initial molar ratioof hydrophobic acid to therapeutic agent (i.e., during formulation ofthe nanoparticles) may be essentially the same as the molar ratio ofhydrophobic acid to therapeutic agent in the nanoparticles (i.e., afterremoval of unencapsulated hydrophobic acid and therapeutic agent).

In an embodiment, when the nanoparticle contains the hydrophobic acid,the nanoparticle comprising the therapeutic agent,1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureamay form a salt with the hydrophobic acid. In other words, thehydrophobic acid associated with the therapeutic agent is thepharmaceutically acceptable salt. Thus, in an embodiment, the presentinvention relates to a therapeutic nanoparticle comprising therapeuticagent or a pharmaceutically acceptable salt thereof and a polymerselected from diblock poly(lactic) acid-poly(ethylene)glycol copolymeror a diblock poly(lactic acid-co-glycolic acid)-poly(ethylene)glycolcopolymer and combination thereof, wherein the therapeutic agent is1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea.

In some cases, a solution containing the therapeutic agent may beprepared separately from a solution containing the polymer, and the twosolutions may then be combined prior to nanoparticle formulation. Forinstance, in one embodiment, a first solution contains the therapeuticagent and the hydrophobic acid, and a second solution contains thepolymer and optionally the hydrophobic acid. Formulations where thesecond solution does not contain the hydrophobic acid may beadvantageous, for example, for minimizing the amount of hydrophobic acidused in a process or, in some cases, for minimizing contact time betweenthe hydrophobic acid and, e.g., a polymer that can degrade in thepresence of the hydrophobic acid. In other cases, a single solution maybe prepared containing the therapeutic agent, polymer, and hydrophobicacid.

In some embodiments, the hydrophobic ion pair may be formed prior toformulation of the nanoparticles. For example, a solution containing thehydrophobic ion pair may be prepared prior to formulating thecontemplated nanoparticles (e.g., by preparing a solution containingsuitable amounts of the therapeutic agent and the hydrophobic acid). Inother embodiments, the hydrophobic ion pair may be formed duringformulation of the nanoparticles. For example, a first solutioncontaining the therapeutic agent and a second solution containing thehydrophobic acid may be combined during a process step for preparing thenanoparticles (e.g., prior to emulsion formation and/or during emulationformation). In certain embodiments, the hydrophobic ion pair may formprior to encapsulation of the therapeutic agent and hydrophobic acid ina contemplated nanoparticle. In other embodiments, the hydrophobic ionpair may form in the nanoparticle, e.g., after encapsulation of thetherapeutic agent and hydrophobic acid.

In certain embodiments, the hydrophobic acid may have a solubility ofless than about 2 g per 100 mL of water or less, in some embodimentsabout 1 g per 100 mL of water or less; in some embodiments, about 100 mgper 100 mL of water or less, in some embodiments, about 10 mg per 100 mLof water or less, and in some embodiments about 1 mg per 100 mL of wateror less, determined at 25° C. In other embodiments, the hydrophobic acidmay have a solubility ranging from about 1 mg per 100 mL of water toabout 2 g per 100 mL of water; in some embodiments from about 1 mg per100 mL of water to about 1 g per 100 mL of water, in some embodiments,from about 1 mg per 100 mL of water to about 500 mg per 100 mL of water,and in some embodiments from about 1 mg per 100 mL of water to about 100mg per 100 mL of water, determined at 25° C. In some embodiments, thehydrophobic acid may be essentially insoluble in water at 25° C.

In certain embodiments, the hydrophobic acid may have a solubility ofless than 2 g per 100 mL of water or less, in some embodiments 1 g per100 mL of water or less; in some embodiments, 100 mg per 100 mL of wateror less, in some embodiments, 10 mg per 100 mL of water or less, and insome embodiments 1 mg per 100 mL of water or less, determined at 25° C.In other embodiments, the hydrophobic acid may have a solubility rangingfrom 1 mg per 100 mL of water to 2 g per 100 mL of water; in someembodiments from 1 mg per 100 mL of water to 1 g per 100 mL of water, insome embodiments, from 1 mg per 100 mL of water to 500 mg per 100 mL ofwater, and in some embodiments from 1 mg per 100 mL of water to 100 mgper 100 mL of water, determined at 25° C. In some embodiments, thehydrophobic acid may be essentially insoluble in water at 25° C.

In some embodiments, disclosed nanoparticles may be essentially free ofthe hydrophobic acid used during the preparation of the nanoparticles.In other embodiments, disclosed nanoparticles may comprise thehydrophobic acid. For instance, in some embodiments, the acid content indisclosed nanoparticles may range from about 0.05 weight percent toabout 35 weight percent, in some embodiments from about 0.05 weightpercent to about 30 weight percent, in some embodiments from about 0.05weight percent to about 20 weight percent, in some embodiments, fromabout 0.5 weight percent to about 30 weight percent, in some embodimentsfrom about 1 weight percent to about 30 weight percent, in someembodiments from about 2 weight percent to about 30 weight percent, insome embodiments from about 3 weight percent to about 30 weight percent,in some embodiments, from about 5 weight percent to about 30 weightpercent, in some embodiments, from about 7 weight percent to about 30weight percent, in some embodiments, from about 10 weight percent toabout 30 weight percent, in some embodiments, from about 15 weightpercent to about 25 weight percent, in some embodiments, from about 15weight percent to about 30 weight percent, in some embodiments, fromabout 20 weight percent to about 30 weight percent, in some embodiments,from about 0.05 weight percent to about 0.5 weight percent, in someembodiments, from about 0.05 weight percent to about 5 weight percent,in some embodiments, from about 1 weight percent to about 5 weightpercent, in some embodiments from about 3 weight percent to about 10weight percent, in some embodiments, from about 1 weight percent toabout 10 weight percent, in some embodiments, from about 5 weightpercent to about 10 weight percent, in some embodiments, from about 5weight percent to about 15 weight percent, and in some embodiments, fromabout 10 weight percent to about 20 weight percent.

In some embodiments, the acid content in disclosed nanoparticles mayrange from 0.05 weight percent to 35 weight percent, in some embodimentsfrom 0.05 weight percent to 30 weight percent, in some embodiments from0.05 weight percent to 20 weight percent, in some embodiments, from 0.5weight percent to 30 weight percent, in some embodiments from 1 weightpercent to 30 weight percent, in some embodiments from 2 weight percentto 30 weight percent, in some embodiments from 3 weight percent to 30weight percent, in some embodiments, from 5 weight percent to 30 weightpercent, in some embodiments, from 7 weight percent to 30 weightpercent, in some embodiments, from 10 weight percent to 30 weightpercent, in some embodiments, from 15 weight percent to 25 weightpercent, in some embodiments, from 15 weight percent to 30 weightpercent, in some embodiments, from 20 weight percent to 30 weightpercent, in some embodiments, from 0.05 weight percent to 0.5 weightpercent, in some embodiments, from 0.05 weight percent to 5 weightpercent, in some embodiments, from 1 weight percent to 5 weight percent,in some embodiments from 3 weight percent to 10 weight percent, in someembodiments, from about 1 weight percent to about 10 weight percent, insome embodiments, from about 5 weight percent to about 10 weightpercent, in some embodiments, from 5 weight percent to 15 weightpercent, and in some embodiments, from 10 weight percent to 20 weightpercent.

In some embodiments, disclosed nanoparticles substantially immediatelyrelease (e.g., from about 1 minute to about 30 minutes, about 1 minuteto about 25 minutes, about 5 minutes to about 30 minutes, about 5minutes to about 1 hour, about 1 hour, or about 24 hours). In othercases, the release profile is slower: about 2% or less; about 5% orless; about 10% or less; about 15% or less; about 20% or less; about 25%or, about 30% or less about 40% or less of the therapeutic agent, byweight is released for example, when placed in a phosphate buffersolution, e.g. a buffer comprising monobasic and dibasic phosphatebuffer (such as 0.138 M sodium chloride, 0.0027 M potassium chloride,about 0.02 M monobasic sodium or potassium phosphate and about 0.01 Msodium or potassium dibasic phosphate buffer dissolved in 1 liter ofwater, e.g., RODI water), at room temperature (e.g., 25° C.) and/or at37° C. In certain embodiments, nanoparticles comprising the therapeuticagent may release the therapeutic agent when placed in an aqueoussolution (e.g., a phosphate buffer solution, such as described hereinabove) e.g., at 25° C. and/or at 37° C., at a rate substantiallycorresponding to about 0.01 to about 50%, in some embodiments about 0.01to about 25%, in some embodiments about 0.01 to about 15%, in someembodiments about 0.01 to about 10%, in some embodiments about 1 toabout 40%, in some embodiments about 5 to about 40%, and in someembodiments about 10 to about 40% of the therapeutic agent released byweight over about 1 hour. In some embodiments, nanoparticles comprisingthe therapeutic agent may release the therapeutic agent when placed inan aqueous solution (e.g., a phosphate buffer solution), e.g., at 25° C.and/or at 37° C., at a rate substantially corresponding to about 10 toabout 70%, in some embodiments about 10 to about 45%, in someembodiments about 10 to about 35%, or in some embodiments about 10 toabout 25%, therapeutic agent, released by weight over about 4 hours. Incertain embodiments, nanoparticles comprising the therapeutic agent mayrelease the therapeutic agent when placed in an aqueous solution (e.g.,a phosphate buffer solution, such as described hereinabove) e.g., at 25°C. and/or at 37° C., at a rate substantially corresponding to about 0.01to about 50%, in some embodiments about 0.01 to about 25%, in someembodiments about 0.01 to about 15%, in some embodiments about 0.01 toabout 10%, in some embodiments about 0.01 to about 5%, and in someembodiments about 0.01 to about 3% of the therapeutic agent released byweight over about 4 hours. In certain embodiments, nanoparticlescomprising the therapeutic agent may release the therapeutic agent whenplaced in an aqueous solution (e.g., a phosphate buffer solution, suchas described hereinabove) e.g., at 25° C. and/or at 37° C., at a ratesubstantially corresponding to about 0.01 to about 60%, in someembodiments about 0.01 to about 25%, in some embodiments about 0.01 toabout 15%, in some embodiments about 0.01 to about 10%, in someembodiments about 0.01 to about 5%, and in some embodiments about 0.01to about 3% of the therapeutic agent released by weight over about 10hours. In certain embodiments, nanoparticles comprising the therapeuticagent may release the therapeutic agent when placed in an aqueoussolution (e.g., a phosphate buffer solution, such as describedhereinabove) e.g., at 25° C. and/or at 37° C., at a rate substantiallycorresponding to about 0.01 to about 70%, in some embodiments about 0.01to about 50%, in some embodiments about 0.01 to about 25%, in someembodiments about 0.01 to about 15%, in some embodiments about 0.01 toabout 10%, in some embodiments about 0.01 to about 5%, and in someembodiments about 0.01 to about 3% of the therapeutic agent released byweight over about 20 hours. In certain embodiments, nanoparticlescomprising the therapeutic agent may release the therapeutic agent whenplaced in an aqueous solution (e.g., a phosphate buffer solution, suchas described hereinabove) e.g., at 25° C. and/or at 37° C., at a ratesubstantially corresponding to about 1 to about 80%, in some embodimentsabout 1 to about 50%, in some embodiments about 1 to about 30%, in someembodiments about 1 to about 25%, in some embodiments about 1 to about15%, in some embodiments about 1 to about 10%, and in some embodimentsabout 1 to about 5% of the therapeutic agent released by weight overabout 40 hours. In certain embodiments, nanoparticles comprising thetherapeutic agent may release the therapeutic agent when placed in anaqueous solution (e.g., a phosphate buffer solution, such as describedhereinabove) e.g., at 25° C. and/or at 37° C., at a rate substantiallycorresponding to about 10 to about 100%, in some embodiments about 10 toabout 80%, in some embodiments about 10 to about 70%, in someembodiments about 10 to about 60%, in some embodiments about 10 to about50%, in some embodiments about 10 to about 40%, in some embodimentsabout 10 to about 30%, in some embodiments about 10 to about 20% of thetherapeutic agent released by weight over about 100 hours.

In some embodiments, disclosed nanoparticles substantially immediatelyrelease (e.g., from 1 minute to 30 minutes, 1 minute to 25 minutes, 5minutes to 30 minutes, 5 minutes to 1 hour, 1 hour, or 24 hours). Inother cases, the release profile is slower: 2% or less; 5% or less; 10%or less; 15% or less; 20% or less; 25% or, 30% or less 40% or less ofthe therapeutic agent, by weight is released for example, when placed ina phosphate buffer solution, e.g. a buffer comprising monobasic anddibasic phosphate buffer (such as 0.138 M sodium chloride, 0.0027 Mpotassium chloride, about 0.02 M monobasic sodium or potassium phosphateand about 0.01 M sodium or potassium dibasic phosphate buffer dissolvedin 1 liter of water, e.g., RODI water), at room temperature (e.g., 25°C.) and/or at 37° C. In certain embodiments, nanoparticles comprisingthe therapeutic agent may release the therapeutic agent when placed inan aqueous solution (e.g., a phosphate buffer solution, such asdescribed hereinabove) e.g., at 25° C. and/or at 37° C., at a ratesubstantially corresponding to 0.01 to 50%, in some embodiments 0.01 to25%, in some embodiments 0.01 to 15%, in some embodiments 0.01 to 10%,in some embodiments 1 to 40%, in some embodiments 5 to 40%, and in someembodiments 10 to 40% of the therapeutic agent released by weight over 1hour. In some embodiments, nanoparticles comprising the therapeuticagent may release the therapeutic agent when placed in an aqueoussolution (e.g., a phosphate buffer solution), e.g., at 25° C. and/or at37° C., at a rate substantially corresponding to 10 to 70%, in someembodiments 10 to 45%, in some embodiments 10 to 35%, or in someembodiments 10 to 25%, therapeutic agent, released by weight over 4hours. In certain embodiments, nanoparticles comprising the therapeuticagent may release the therapeutic agent when placed in an aqueoussolution (e.g., a phosphate buffer solution, such as describedhereinabove) e.g., at 25° C. and/or at 37° C., at a rate substantiallycorresponding to 0.01 to 50%, in some embodiments 0.01 to 25%, in someembodiments 0.01 to 15%, in some embodiments 0.01 to 10%, in someembodiments 0.01 to 5%, and in some embodiments 0.01 to 3% of thetherapeutic agent released by weight over 4 hours. In certainembodiments, nanoparticles comprising the therapeutic agent may releasethe therapeutic agent when placed in an aqueous solution (e.g., aphosphate buffer solution, such as described hereinabove) e.g., at 25°C. and/or at 37° C., at a rate substantially corresponding to 0.01 to60%, in some embodiments 0.01 to 25%, in some embodiments 0.01 to 15%,in some embodiments 0.01 to 10%, in some embodiments 0.01 to 5%, and insome embodiments 0.01 to 3% of the therapeutic agent released by weightover 10 hours. In certain embodiments, nanoparticles comprising thetherapeutic agent may release the therapeutic agent when placed in anaqueous solution (e.g., a phosphate buffer solution, such as describedhereinabove) e.g., at 25° C. and/or at 37° C., at a rate substantiallycorresponding to 0.01 to 70%, in some embodiments 0.01 to 50%, in someembodiments 0.01 to 25%, in some embodiments 0.01 to 15%, in someembodiments 0.01 to 10%, in some embodiments 0.01 to 5%, and in someembodiments 0.01 to 3% of the therapeutic agent released by weight over20 hours. In certain embodiments, nanoparticles comprising thetherapeutic agent may release the therapeutic agent when placed in anaqueous solution (e.g., a phosphate buffer solution, such as describedhereinabove) e.g., at 25° C. and/or at 37° C., at a rate substantiallycorresponding 1 to 80%, in some embodiments 1 to 50%, in someembodiments 1 to 30%, in some embodiments 1 to 25%, in some embodiments1 to 15%, in some embodiments 1 to 10%, and in some embodiments 1 to 5%of the therapeutic agent released by weight over 40 hours. In certainembodiments, nanoparticles comprising the therapeutic agent may releasethe therapeutic agent when placed in an aqueous solution (e.g., aphosphate buffer solution, such as described hereinabove) e.g., at 25°C. and/or at 37° C., at a rate substantially corresponding to 10 to100%, in some embodiments 10 to 80%, in some embodiments 10 to 70%, insome embodiments 10 to 60%, in some embodiments 10 to 50%, in someembodiments 10 to 40%, in some embodiments 10 to 30%, in someembodiments 10 to 20% of the therapeutic agent released by weight over100 hours.

In some embodiments, disclosed nanoparticles may substantially retainthe therapeutic agent, e.g., for at least about 1 minute, at least about1 hour, or more, when placed in a phosphate buffer solution at 37° C.

In some embodiments, disclosed nanoparticles may substantially retainthe therapeutic agent, e.g., for at least 1 minute, at least 1 hour, ormore, when placed in a phosphate buffer solution at 37° C.

In one embodiment, disclosed therapeutic nanoparticles may include atargeting ligand, e.g., a low-molecular weight ligand. In certainembodiments, the low-molecular weight ligand is conjugated to a polymer,and the nanoparticle comprises a certain ratio of ligand-conjugatedpolymer (e.g., PLA-PEG-Ligand) to non-functionalized polymer (e.g.,PLA-PEG or PLGA-PEG). The nanoparticle can have an effective ratio ofthese two polymers such that an effective amount of ligand is associatedwith the nanoparticle for treatment of a disease or disorder, such ascancer. For example, an increased ligand density may increase targetbinding (cell binding/target uptake), making the nanoparticle “targetspecific.” Alternatively, a certain concentration of non-functionalizedpolymer (e.g., non-functionalized PLGA-PEG copolymer) in thenanoparticle can control inflammation and/or immunogenicity (i.e., theability to provoke an immune response), and allow the nanoparticle tohave a circulation half-life that is adequate for the treatment of adisease or disorder. For instance, in an embodiment, the molar ratio ofnon-functionalized polymer to the ligand conjugated polymer ranges fromabout 0.01 to about 0.1 and in another embodiment, from about 0.01 toabout 0.05, such as, e.g. about 0.025. Furthermore, thenon-functionalized polymer may, in some embodiments, lower the rate ofclearance from the circulatory system via the reticuloendothelial system(RES). Thus, the non-functionalized polymer may provide the nanoparticlewith characteristics that may allow the particle to travel through thebody upon administration. In some embodiments, a non-functionalizedpolymer may balance an otherwise high concentration of ligands, whichcan otherwise accelerate clearance by the subject, resulting in lessdelivery to the target cells.

In another embodiment, the molar ratio of non-functionalized polymer tothe ligand conjugated polymer ranges from 0.01 to 0.1 and in anotherembodiment, from 0.01 to 0.05, such as, e.g. 0.025.

In some embodiments, nanoparticles disclosed herein may includefunctionalized polymers conjugated to a ligand that constitute a rangefrom approximately 0.1 to about 50. e.g., about 0.1 to about 30, e.g.,about 0.1 to about 20, e.g., about 0.1 to about 10 mole percent of theentire polymer composition of the nanoparticle (i.e.,functionalized+non-functionalized polymer). Also disclosed herein, inanother embodiment, are nanoparticles that include a polymer conjugated(e.g., covalently with (i.e., through a linker (e.g., an alkylenelinker)) or a bond) with one or more low-molecular weight ligands,wherein the weight percent low-molecular weight ligand with respect tototal polymer is ranges from about 0.001 to about 5, e.g., from about0.001 to about 2, e.g., from and about 0.001 to about 1.

In some embodiments, nanoparticles disclosed herein may includefunctionalized polymers conjugated to a ligand that constitute a rangefrom 0.1-50, e.g., 0.1-30, e.g., 0.1-20, e.g., 0.1-10 mole percent ofthe entire polymer composition of the nanoparticle (i.e.,functionalized+non-functionalized polymer). Also disclosed herein arenanoparticles that include a polymer conjugated with one or morelow-molecular weight ligands, wherein the weight percent low-molecularweight ligand with respect to total polymer is ranges from 0.001 to 5,e.g., from 0.001 to 2, e.g., from and 0.001 to 1.

In general, a “nanoparticle” refers to any particle having a diameter ofless than 1000 nm, e.g., about 10 nm to about 200 nm. Disclosedtherapeutic nanoparticles may include nanoparticles having a diameterranging from about 60 to about 120 nm, or from about 70 to about 120 nm,or from about 80 to about 120 nm, or from about 90 to about 120 nm, orfrom about 100 to about 120 nm, or from about 60 to about 130 nm, orfrom about 70 to about 130 nm, or from about 80 to about 130 nm, or fromabout 90 to about 130 nm, or from about 100 to about 130 nm, or fromabout 110 to about 130 nm, or from about 60 to about 140 nm, or fromabout 70 to about 140 nm, or from about 80 to about 140 nm, or fromabout 90 to about 140 nm, or from about 100 to about 140 nm, or fromabout 110 to about 140 nm, or from about 60 to about 150 nm, or fromabout 70 to about 150 nm, or from about 80 to about 150 nm, or fromabout 90 to about 150 nm, or from about 100 to about 150 nm, or fromabout 110 to about 150 nm, or from about 120 to about 150 nm.

Disclosed therapeutic nanoparticles may include nanoparticles having adiameter ranging from 60 to 120 nm, or from 70 to 120 nm, or from 80 to120 nm, or from 90 to 120 nm, or from 100 to 120 nm, or from 60 to 130nm, or from 70 to 130 nm, or from 80 to 130 nm, or from 90 to 130 nm, orfrom 100 to 130 nm, or from 110 to 130 nm, or from 60 to 140 nm, or from70 to 140 nm, or from 80 to 140 nm, or from 90 to 140 nm, or from 100 to140 nm, or from 110 to 140 nm, or from 60 to 150 nm, or from 70 to 150nm, or from 80 to 150 nm, or from 90 to 150 nm, or from 100 to 150 nm,or from 110 to 150 nm, or from 120 to 150 nm.

Polymers

In some embodiments, the nanoparticles may comprise a matrix of polymersand the therapeutic agent. In some embodiments, the therapeutic agentand/or targeting moiety (i.e., a low-molecular weight ligand) can beassociated with at least part of the polymeric matrix. For example, insome embodiments, a targeting moiety (e.g., ligand) can be covalentlyassociated with the surface of a polymeric matrix. In some embodiments,covalent association is mediated by a linker. The therapeutic agent canbe associated with the surface of, encapsulated within, surrounded by,and/or dispersed throughout the polymeric matrix.

A wide variety of polymers and methods for forming particles therefromare known in the art of drug delivery. In some embodiments, thedisclosure is directed toward nanoparticles with at least twomacromolecules, wherein the first macromolecule comprises a firstpolymer bound to a low-molecular weight ligand (e.g., targeting moiety);and the second macromolecule comprising a second polymer that is notbound to a targeting moiety. The nanoparticle can optionally include oneor more additional, non-non-functionalized, polymers.

Any suitable polymer can be used in the disclosed nanoparticles.Polymers can be natural or unnatural (synthetic) polymers. Polymers canbe homopolymers or copolymers comprising two or more monomers. In termsof sequence, copolymers can be random, block, or comprise a combinationof random and block sequences. Typically, polymers are organic polymers.

The term “polymer,” as used herein, is given its ordinary meaning asused in the art, i.e., a molecular structure comprising one or morerepeat units (monomers), connected by covalent bonds. The repeat unitsmay all be identical, or in some cases, there may be more than one typeof repeat unit present within the polymer. In some cases, the polymercan be biologically derived, i.e., a biopolymer. Non-limiting examplesinclude peptides or proteins. In some cases, additional moieties mayalso be present in the polymer, for example, biological moieties such asthose described below. If more than one type of repeat unit is presentwithin the polymer, then the polymer is said to be a “copolymer.” It isto be understood that in any embodiment employing a polymer, the polymerbeing employed may be a copolymer in some cases. The repeat unitsforming the copolymer may be arranged in any fashion. For example, therepeat units may be arranged in a random order, in an alternating order,or as a block copolymer, i.e., comprising one or more regions eachcomprising a first repeat unit (e.g., a first block), and one or moreregions each comprising a second repeat unit (e.g., a second block),etc. Block copolymers may have two (a diblock copolymer), three (atriblock copolymer), or more numbers of distinct blocks.

Disclosed particles can include copolymers, which, in some embodiments,describe two or more polymers (such as those described herein) that havebeen associated with each other, usually by covalent bonding of the twoor more polymers together. Thus, a copolymer may comprise a firstpolymer and a second polymer, which have been conjugated together toform a block copolymer where the first polymer can be a first block ofthe block copolymer and the second polymer can be a second block of theblock copolymer. Of course, those of ordinary skill in the art willunderstand that a block copolymer may, in some cases, contain multipleblocks of polymer, and that a “block copolymer,” as used herein, is notlimited to only block copolymers having only a single first block and asingle second block. For instance, a block copolymer may comprise afirst block comprising a first polymer, a second block comprising asecond polymer, and a third block comprising a third polymer or thefirst polymer, etc. In some cases, block copolymers can contain anynumber of first blocks of a first polymer and second blocks of a secondpolymer (and in certain cases, third blocks, fourth blocks, etc.). Inaddition, it should be noted that block copolymers can also be formed,in some instances, from other block copolymers. For example, a firstblock copolymer may be conjugated to another polymer (which may be ahomopolymer, a biopolymer, another block copolymer, etc.), to form a newblock copolymer containing multiple types of blocks, and/or to othermoieties (e.g., to nonpolymeric moieties).

In some embodiments, the polymer (e.g., copolymer, e.g., blockcopolymer) can be amphiphilic, i.e., having a hydrophilic portion and ahydrophobic portion, or a relatively hydrophilic portion and arelatively hydrophobic portion. A hydrophilic polymer can be onegenerally that attracts water and a hydrophobic polymer can be one thatgenerally repels water. A hydrophilic or a hydrophobic polymer can beidentified, for example, by preparing a sample of the polymer andmeasuring its contact angle with water (typically, the polymer will havea contact angle of less than 60°, while a hydrophobic polymer will havea contact angle of greater than about 60°). In some cases, thehydrophilicity of two or more polymers may be measured relative to eachother, i.e., a first polymer may be more hydrophilic than a secondpolymer. For instance, the first polymer may have a smaller contactangle than the second polymer.

In one set of embodiments, a polymer (e.g., copolymer, e.g., blockcopolymer) contemplated herein includes a biocompatible polymer, i.e.,the polymer that does not typically induce an adverse response wheninserted or injected into a living subject, for example, withoutsignificant inflammation and/or acute rejection of the polymer by theimmune system, for instance, via a T-cell response. Accordingly, thetherapeutic particles contemplated herein can be non-immunogenic. Theterm non-immunogenic as used herein refers to endogenous growth factorin its native state which normally elicits no, or only minimal levelsof, circulating antibodies, T-cells, or reactive immune cells, and whichnormally does not elicit in the individual an immune response againstitself.

Biocompatibility typically refers to the acute rejection of material byat least a portion of the immune system, i.e., a non-biocompatiblematerial implanted into a subject provokes an immune response in thesubject that can be severe enough such that the rejection of thematerial by the immune system cannot be adequately controlled, and oftenis of a degree such that the material must be removed from the subject.One simple test to determine biocompatibility can be to expose a polymerto cells in vitro; biocompatible polymers are polymers that typicallywill not result in significant cell death at moderate concentrations,e.g., at concentrations of 50 micrograms/10⁶ cells. For instance, abiocompatible polymer may cause less than about 20% cell death whenexposed to cells such as fibroblasts or epithelial cells, even ifphagocytosed or otherwise taken up by such cells. Non-limiting examplesof biocompatible polymers that may be useful in various embodimentsinclude polydioxanone (PDO), polyhydroxyalkanoate, polyhydroxybutyrate,poly(glycerol sebacate), polyglycolide (i.e., poly(glycolic) acid)(PGA), polylactide (i.e., poly(lactic) acid) (PLA), poly(lactic)acid-co-poly(glycolic) acid (PLGA), polycaprolactone, or copolymers orderivatives including these and/or other polymers.

In certain embodiments, contemplated biocompatible polymers may bebiodegradable, i.e., the polymer is able to degrade, chemically and/orbiologically, within a physiological environment, such as within thebody. As used herein, “biodegradable” polymers are those that, whenintroduced into cells, are broken down by the cellular machinery(biologically degradable) and/or by a chemical process, such ashydrolysis, (chemically degradable) into components that the cells caneither reuse or dispose of without significant toxic effect on thecells. In one embodiment, the biodegradable polymer and theirdegradation byproducts can be biocompatible.

Particles disclosed herein may or may not contain PEG. In addition,certain embodiments can be directed towards copolymers containingpoly(ester-ether)s, e.g., polymers having repeat units joined by esterbonds (e.g., R¹⁰⁰—C(O)—O—R¹ bonds) and ether bonds (e.g., R¹—O—R¹ bondswherein R¹⁰⁰ and R¹ are independently hydrocarbyl moieties which mayoptionally be substituted and which may be the same or different). Insome embodiments, a biodegradable polymer, such as a hydrolyzablepolymer, containing carboxylic acid groups, may be conjugated withpoly(ethylene glycol) repeat units to form a poly(ester-ether). Apolymer (e.g., copolymer, e.g., block copolymer) containingpoly(ethylene glycol) repeat units can also be referred to as a“PEGylated” polymer.

For instance, a contemplated polymer may be one that hydrolyzesspontaneously upon exposure to water (e.g., within a subject), or thepolymer may degrade upon exposure to heat (e.g., at temperatures ofabout 37° C.). Degradation of a polymer may occur at varying rates,depending on the polymer or copolymer used. For example, the half-lifeof the polymer (the time at which 50% of the polymer can be degradedinto monomers and/or other nonpolymeric moieties) may be on the order ofdays, weeks, months, or years, depending on the polymer. The polymersmay be biologically degraded, e.g., by enzymatic activity or cellularmachinery, in some cases, for example, through exposure to a lysozyme(e.g., having relatively low pH). In some cases, the polymers may bebroken down into monomers and/or other nonpolymeric moieties that cellscan either reuse or dispose of without significant toxic effect on thecells (for example, polylactide may be hydrolyzed to form lactic acid,polyglycolide may be hydrolyzed to form glycolic acid, etc.).

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEGylated polymers andcopolymers of lactide and glycolide (e.g., PEGylated PLA, PEGylated PGA,PEGylated PLGA, and derivatives thereof). In some embodiments,polyesters include, for example, polyanhydrides, poly(ortho ester)PEGylated poly(ortho ester), poly(caprolactone), PEGylatedpoly(caprolactone), polylysine, PEGylated polylysine, poly(ethyleneimine), PEGylated poly(ethylene imine), poly(L-lactide-co-L-lysine),poly(serine ester), poly(4-hydroxy-L-proline ester),poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA can be characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid-glycolic acid ratio. In some embodiments. PLGA can becharacterized by a lactic acid:glycolic acid molar ratio ofapproximately 85:15, approximately 75:25, approximately 60:40,approximately 50:50, approximately 40:60, approximately 25:75, orapproximately 15:85. In some embodiments, the molar ratio of lactic acidto glycolic acid monomers in the polymer of the particle (e.g., the PLGAblock copolymer or PLGA-PEG block copolymer), may be selected tooptimize for various parameters such as water uptake, therapeutic agentrelease and/or polymer degradation kinetics can be optimized.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid polyacrylamide, amino alkyl methacrylatecopolymer, glycidyl methacrylate copolymers, polycyanoacrylates, andcombinations comprising one or more of the foregoing polymers. Theacrylic polymer may comprise fully-polymerized copolymers of acrylic andmethacrylic acid esters with a low content of quaternary ammoniumgroups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g., DNA, RNA, or derivatives thereof).Amine-containing polymers such as poly(lysine), polyethylene imine(PEI), and poly(amidoamine) dendrimers are contemplated for use, in someembodiments, in a disclosed particle.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains. Examples of these polyesters includepoly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester).

It is contemplated that PEG may be terminated and include an end group,for example, when PEG is not conjugated to a ligand. For example, PEGmay terminate in a hydroxyl, a methoxy or other alkoxyl group, a methylor other alkyl group, an aryl group, a carboxylic acid, an amine, anamide, an acetyl group, a guanidino group, or an imidazole. Othercontemplated end groups include azide, alkyne, maleimide, aldehyde,hydrazide, hydroxylamine, alkoxyamine, or thiol moieties.

Those of ordinary skill in the art will know of methods and techniquesfor PEGylating a polymer, for example, by using EDC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS(N-hydroxysuccinimide) to react a polymer to a PEG group terminating inan amine, by ring opening polymerization techniques (ROMP), or the like.

In one embodiment, the molecular weight (or e.g., the ratio of molecularweights of, e.g., different blocks of a copolymer) of the polymers canbe optimized for effective treatment as disclosed herein. For example,the molecular weight of a polymer may influence particle degradationrate (such as when the molecular weight of a biodegradable polymer canbe adjusted), solubility, water uptake, and drug release kinetics. Forexample, the molecular weight of the polymer (or e.g., the ratio ofmolecular weights of, e.g., different blocks of a copolymer) can beadjusted such that the particle biodegrades in the subject being treatedwithin a reasonable period of time (ranging from a few hours to 1-2weeks, 3-4 weeks, 5-6 weeks, 7-8 weeks, etc.).

A disclosed particle can for example comprise a diblock copolymer of PEGand PL(G)A, wherein for example, the PEG portion may have a numberaverage molecular weight of about 1,000-20,000, e.g., about2,000-20,000, e.g., about 2 to about 10,000, and the PL(G)A portion mayhave a number average molecular weight of about 5,000 to about 20,000,or about 5,000-100,000, e.g., about 20,000-70,000, e.g., about15,000-50,000.

For example, disclosed here is an exemplary therapeutic nanoparticlethat includes from about 10 to about 99 weight percent poly(lactic)acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly(glycolic)acid-poly(ethylene)glycol copolymer, or from about 50 to about 99.75weight percent, from about 20 to about 80 weight percent, from about 40to about 80 weight percent, or from about 30 to about 50 weight percent,or from about 70 to about 90 weight percent, from about 70 to about99.75 weight percent, from about 80 to about 99.75 weight percent, fromabout 70 to about 80 weight percent, or from about 85 to about 95 weightpercent poly(lactic) acid-poly(ethylene)glycol copolymer orpoly(lactic)-co-poly(glycolic) acid-poly(ethylene)glycol copolymer. Insome embodiments, a therapeutic nanoparticle comprises about 50 weightpercent, about 55 weight percent, about 60 weight percent, about 65weight percent, about 70 weight percent, about 75 weight percent, about80 weight percent, about 85 weight percent, about 90 weight percent orabout 95 weight percent poly(lactic) acid-poly(ethylene)glycol copolymeror poly(lactic)-co-poly(glycolic) acid-poly(ethylene)glycol copolymer.Exemplary poly(lactic) acid-poly(ethylene)glycol copolymers can includea number average molecular weight ranging from about 15 to about 20 kDa,or from about 10 to about 25 kDa of poly(lactic) acid and a numberaverage molecular weight from about 4 kDa to about 6 kDa, from about 4kDa to about 10 kD, from about 6 kDa to about 10 kDa, or from about 2kDa to about 10 kDa of poly(ethylene)glycol.

In another example, disclosed here is an exemplary therapeuticnanoparticle that includes from 10 to 99 weight percent poly(lactic)acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly(glycolic)acid-poly(ethylene)glycol copolymer, or from 50 to 99.75 weight percent,from 20 to 80 weight percent, from 40 to 80 weight percent, or from 30to 50 weight percent, or from 70 to 90 weight percent, from 70 to 99.75weight percent, from 80 to 99.75 weight percent, from 70 to 80 weightpercent, or from 85 to 95 weight percent poly(lactic)acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly(glycolic)acid-poly(ethylene)glycol copolymer. In some embodiments, a therapeuticnanoparticle comprises 50 weight percent, 55 weight percent, 60 weightpercent, 65 weight percent, 70 weight percent, 75 weight percent, 80weight percent, 85 weight percent, 90 weight percent or 95 weightpercent poly(lactic) acid-poly(ethylene)glycol copolymer orpoly(lactic)-co-poly(glycolic) acid-poly(ethylene)glycol copolymer.Exemplary poly(lactic) acid-poly(ethylene)glycol copolymers can includea number average molecular weight ranging from 15 to 20 kDa, or from 10to 25 kDa of poly(lactic) acid and a number average molecular weightfrom 4 kDa to 6 kDa, from 4 kDa to 10 kD, from 6 kDa to 10 kDa, or from2 kDa to 10 kDa of poly(ethylene)glycol.

In some embodiments, the poly(lactic) acid-poly(ethylene)glycolcopolymer may have a poly(lactic) acid number average molecular weightfraction of from about 0.6 to about 0.95, in some embodiments, fromabout 0.7 to about 0.9, in some embodiments, from about 0.6 to about0.8, in some embodiments, from about 0.7 to about 0.8, in someembodiments, from about 0.75 to about 0.85, in some embodiments fromabout 0.8 to about 0.9, and in some embodiments, from about 0.85 toabout 0.95. It should be understood that the poly(lactic) acid numberaverage molecular weight fraction may be calculated by dividing thenumber average molecular weight of the poly(lactic) acid component ofthe copolymer by the sum of the number average molecular weight of thepoly(lactic) acid component and the number average molecular weight ofthe poly(ethylene)glycol component.

In some embodiments, the poly(lactic) acid-poly(ethylene)glycolcopolymer may have a poly(lactic) acid number average molecular weightfraction of from 0.6 to 0.95, in some embodiments, from 0.7 to 0.9, insome embodiments, from 0.6 to 0.8, in some embodiments, from 0.7 to 0.8,in some embodiments, from 0.75 to 0.85, in some embodiments from 0.8 to0.9, and in some embodiments, from 0.85 to 0.95.

In certain embodiments, the therapeutic nanoparticle comprises1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand PLA-PEG (in a 16:5 molar ratio) in a weight ratio of about 1:7. Incertain embodiments, the therapeutic nanoparticle comprises1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand PLA-PEG (in a 16:5 molar ratio) in a weight ratio of about 1:4. Incertain embodiments, the therapeutic nanoparticle comprises1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand PLA-PEG (in a 16:5 molar ratio) in a weight ratio of about 1:14. Incertain embodiments, the therapeutic nanoparticle comprises1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand PLA-PEG (in a 16:5 molar ratio) in a weight ratio of about 1:3.

In certain embodiments, the therapeutic nanoparticle comprises1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand PLA-PEG (in a 16:5 molar ratio) in a weight ratio of 1:7. In certainembodiments, the therapeutic nanoparticle comprises1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand PLA-PEG (in a 16:5 molar ratio) in a weight ratio of 1:4. In certainembodiments, the therapeutic nanoparticle comprises1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand PLA-PEG (in a 16:5 molar ratio) in a weight ratio of 1:14. Incertain embodiments, the therapeutic nanoparticle comprises1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand PLA-PEG (in a 16:5 molar ratio) in a weight ratio of 1:3.

Disclosed nanoparticles may optionally include about 1 to about 50weight percent poly(lactic) acid or poly(lactic) acid-co-poly(glycolic)acid (which does not include PEG), or may optionally include from about1 to about 50 weight percent, or from about 10 to about 50 weightpercent or from about 30 to about 50 weight percent poly(lactic) acid orpoly(lactic) acid-co-poly(glycolic) acid. For example, poly(lactic) orpoly(lactic)-co-poly(glycolic) acid may have a number average moleculeweight ranging from about 5 to about 15 kDa, or from about 5 to about 12kDa. Exemplary PLA may have a number average molecular weight rangingfrom about 5 to about 10 kDa. Exemplary PLGA may have a number averagemolecular weight ranging from about 8 to about 12 kDa.

Disclosed nanoparticles may optionally include 1 to 50 weight percentpoly(lactic) acid or poly(lactic) acid-co-poly(glycolic) acid (whichdoes not include PEG), or may optionally include from 1 to 50 weightpercent, or from 10 to 50 weight percent or from 30 to 50 weight percentpoly(lactic) acid or poly(lactic) acid-co-poly(glycolic) acid. Forexample, poly(lactic) or poly(lactic)-co-poly(glycolic) acid may have anumber average molecule weight ranging from 5 to 15 kDa, or from 5 to 12kDa. Exemplary PLA may have a number average molecular weight rangingfrom 5 to 10 kDa. Exemplary PLGA may have a number average molecularweight ranging from 8 to 12 kDa.

A therapeutic nanoparticle may, in some embodiments, contain from about10 to about 30 weight percent, from about 10 to about 25 weight percent,from about 10 to about 20 weight percent, from about 10 to about 15weight percent, from about 15 to about 20 weight percent, from about 15to about 25 weight percent, from about 20 to about 25 weight percent,from about 20 to about 30 weight percent, or from about 25 to about 30weight percent of poly(ethylene)glycol, where the poly(ethylene)glycolmay be present as a poly(lactic) acid-poly(ethylene)glycol copolymer,poly(lactic)-co-poly(glycolic) acid-poly(ethylene)glycol copolymer, orpoly(ethylene)glycol homopolymer. In certain embodiments, the polymersof the nanoparticles can be conjugated to a lipid. The polymer can be,for example, a lipid-terminated PEG.

A therapeutic nanoparticle may, in some embodiments, contain from 10 to30 weight percent, from 10 to 25 weight percent, from 10 to 20 weightpercent, from 10 to 15 weight percent, from 15 to 20 weight percent,from 15 to 25 weight percent, from 20 to 25 weight percent, from 20 to30 weight percent, or from 25 to 30 weight percent ofpoly(ethylene)glycol, where the poly(ethylene)glycol may be present as apoly(lactic) acid-poly(ethylene)glycol copolymer,poly(lactic)-co-poly(glycolic) acid-poly(ethylene)glycol copolymer, orpoly(ethylene)glycol homopolymer.

In certain embodiments, the therapeutic nanoparticle comprises thepolymer PLA-PEG and the mole ratio of PLA-PEG is about 5:1. In otherembodiments, the therapeutic nanoparticle comprises the polymer PLA-PEGand the mole ratio of PLA-PEG is 5:1.

Targeting Moieties

Provided herein, in some embodiments, are nanoparticles that may includean optional targeting moiety, i.e., a moiety able to bind to orotherwise associate with a biological entity, for example, a membranecomponent, a cell surface receptor, an antigen, or the like. A targetingmoiety present on the surface of the particle may allow the particle tobecome localized at a particular targeting site, for instance, a tumor,a disease site, a tissue, an organ, a type of cell, etc. As such, thenanoparticle may then be “target specific.” The drug or other payloadmay then, in some cases, be released from the particle and allowed tointeract locally with the particular targeting site.

In one embodiment, a disclosed nanoparticle includes a targeting moietythat is a low-molecular weight ligand. The term “bind” or “binding,” asused herein, refers to the interaction between a corresponding pair ofmolecules or portions thereof that exhibit mutual affinity or bindingcapacity, typically due to specific or non-specific binding orinteraction, including, but not limited to, biochemical, physiological,and/or chemical interactions. “Biological binding” defines a type ofinteraction that occurs between pairs of molecules including proteins,nucleic acids, glycoproteins, carbohydrates, hormones, or the like. Theterm “binding partner” refers to a molecule that can undergo bindingwith a particular molecule. “Specific binding” refers to molecules, suchas polynucleotides, that are able to bind to or recognize a bindingpartner (or a limited number of binding partners) to a substantiallyhigher degree than to other, similar biological entities. In one set ofembodiments, the targeting moiety has an affinity (as measured via adisassociation constant) of less than about 1 micromolar, at least about10 micromolar, or at least about 100 micromolar.

In some embodiments, the targeting moiety has an affinity (as measuredvia a disassociation constant) of less than 1 micromolar, at least 10micromolar, or at least 100 micromolar.

For example, a targeting portion may cause the particles to becomelocalized to a tumor (e.g., a solid tumor), a disease site, a tissue, anorgan, a type of cell, etc. within the body of a subject, depending onthe targeting moiety used. For example, a low-molecular weight ligandmay become localized to a solid tumor, e.g., breast or prostate tumorsor cancer cells. The subject may be a human or non-human animal.Examples of subjects include, but are not limited to, a mammal such as adog, a cat, a horse, a donkey, a rabbit, a cow, a pig, a sheep, a goat,a rat, a mouse, a guinea pig, a hamster, a primate, a human or the like.

Contemplated targeting moieties may include small molecules. In certainembodiments, the term “small molecule” refers to organic compounds,whether naturally-occurring or artificially created (e.g., via chemicalsynthesis) that have relatively low molecular weight and that are notproteins, polypeptides, or nucleic acids. Small molecules typically havemultiple carbon-carbon bonds. In certain embodiments, small moleculesare about 2000 g/mol or less in size. In some embodiments, smallmolecules are about 1500 g/mol or less, or about 1000 g/mol or less. Insome embodiments, small molecules are about 800 g/mol or less, 500 g/molor less, for example from about 100 g/mol to about 600 g/mol, or fromabout 200 g/mol to about 500 g/mol.

In certain embodiments, small molecules are 2000 g/mol or less in size.In some embodiments, small molecules are 1500 g/mol or less, or 1000g/mol or less. In some embodiments, small molecules are 800 g/mol orless, 500 g/mol or less, for example from 100 g/mol to 600 g/mol, orfrom 200 g/mol to 500 g/mol.

In some embodiments, the low-molecular weight ligand is of the FormulaeI, II, III or IV:

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof;

wherein m and n are each, independently, 0, 1, 2 or 3; p is 0 or 1;

R¹, R², R⁴, and R⁵ are each, independently, selected from the groupconsisting of substituted or unsubstituted alkyl (e.g., C₁₋₁₀-alkyl,C₁₋₆-alkyl, or C₁₋₄-alkyl), substituted or unsubstituted aryl (e.g.,phenyl or pyridinyl), and any combination thereof; and R³ is H orC₁₋₆-alkyl (e.g., CH₃).

For compounds of Formulae I, II, III and IV, R¹, R², R⁴ or R⁵ comprisepoints of attachment to the nanoparticle, e.g., a point of attachment toa polymer that forms part of a disclosed nanoparticle, e.g., PEG. Thepoint of attachment may be formed by a covalent bond, ionic bond,hydrogen bond, a bond formed by adsorption including chemical adsorptionand physical adsorption, a bond formed from van der Waals bonds, ordispersion forces. For example, if R¹, R², R⁴, or R⁵ are defined as ananiline or C₁₋₆-alkyl-NH₂ group, any hydrogen (e.g., an amino hydrogen)of these functional groups could be removed such that the low-molecularweight ligand is covalently bound to the polymeric matrix (e.g., thePEG-block of the polymeric matrix) of the nanoparticle. As used herein,the term “covalent bond” refers to a bond between two atoms formed bysharing at least one pair of electrons. In particular embodiments of theFormulae I, II, III or IV, R¹, R², R⁴, and R⁵ are each, independently,C₁₋₆-alkyl or phenyl, or any combination of C₁₋₆-alkyl or phenyl, whichare independently substituted one or more times with OH, SH, NH₂, orCO₂H, and wherein the alkyl group may be interrupted by N(H), S, or O.In another embodiment, R¹, R², R⁴, and R⁵ are each, independently,CH₂-Ph, (CH₂)₂—SH, CH₂—SH, (CH₂)₂C(H)(NH₂)CO₂H, CH₂C(H)(NH₂)CO₂H,CH(NH₂)CH₂CO₂H, (CH₂)₂C(H)(SH)CO₂H, CH₂—N(H)-Ph, O—CH₂-Ph, orO—(CH₂)₂-Ph, wherein Ph is phenyl, and wherein each Ph may beindependently substituted one or more times with OH, NH₂, CO₂H, or SH.For these formulae, the NH₂, OH or SH groups serve as the point ofcovalent attachment to the nanoparticle (e.g., —N(H)—PEG, —O-PEG, or—S-PEG).

Exemplary ligands include:

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof, wherein the NH₂, OH, or SH groups serve as the pointof covalent attachment to the nanoparticle (e.g., —N(H)—PEG, —O-PEG, or—S-PEG) or

indicates the point of attachment to the nanoparticle, wherein n is 1,2, 3, 4, 5, or 6, and wherein R is independently selected from the groupconsisting of NH₂, SH, OH, CO₂H, C₁₋₆-alkyl that is substituted withNH₂, SH, OH, or CO₂H, and phenyl that is substituted with NH₂, SH, OH,or CO₂H, and wherein R serves as the point of covalent attachment to thenanoparticle (e.g., —N(H)—PEG, —S-PEG, —O-PEG, or CO₂—PEG). Thesecompounds may be further substituted with NH₂, SH, OH, CO₂H, C₁₋₆-alkylthat is substituted with NH₂, SH, OH, or CO₂H, or phenyl that issubstituted with NH₂, SH, OH or CO₂H, wherein these functional groupscan also serve as the point of covalent attachment to the nanoparticle.

In some embodiments, small molecule targeting moieties that may be usedto target cells associated with solid tumors such as prostate or breastcancer tumors include PSMA peptidase inhibitors such as 2-PMPA, GPI5232,VA-033, phenylalkylphosphonamidates and/or analogs and derivativesthereof. In some embodiments, small molecule targeting moieties that maybe used to target cells associated with prostate cancer tumors includethiol and indole thiol derivatives, such as 2-MPPA and3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid derivatives. In someembodiments, small molecule targeting moieties that may be used totarget cells associated with prostate cancer tumors include hydroxamatederivatives. In some embodiments, small molecule targeting moieties thatmay be used to target cells associated with prostate cancer tumorsinclude PBDA- and urea-based inhibitors, such as ZJ 43, ZJ 11, ZJ 17, ZJ38 and/or and analogs and derivatives thereof, androgen receptortargeting agents (ARTAs), polyamines, such as putrescine, spermine, andspermidine, inhibitors of the enzyme glutamate carboxylase II (GCPII),also known as NAAG Peptidase or NAALADase.

In another embodiment, the targeting moiety can be a ligand that targetsHer2, EGFR, folate receptor or toll receptors. In another embodiment,the targeting moiety is folate, folic acid, or an EGFR binding molecule.

For example, contemplated the targeting moieties may include a nucleicacid, polypeptide, glycoprotein, carbohydrate, or lipid. For example, atargeting moiety can be a nucleic acid targeting moiety (e.g. anaptamer, e.g., the A10 aptamer) that binds to a cell type specificmarker. In general, an aptamer is an oligonucleotide (e.g., DNA, RNA, oran analog or derivative thereof) that binds to a particular target, suchas a polypeptide. In some embodiments, a targeting moiety may be anaturally occurring or synthetic ligand for a cell surface receptor,e.g., a growth factor, hormone, LDL, transferrin, etc. A targetingmoiety can be an antibody, which term is intended to include antibodyfragments. Characteristic portions of antibodies, single chain targetingmoieties can be identified, e.g., using procedures such as phagedisplay.

Targeting moieties disclosed herein can be, in some embodiments,conjugated to a disclosed polymer or copolymer (e.g., PLA-PEG), and sucha polymer conjugate may form part of a disclosed nanoparticle.

In certain embodiments, the therapeutic nanoparticle has a targetingligand additionally present and the ligand is PLA-PEG-GL, wherein GL hasthe following structure:

In some embodiments, a therapeutic nanoparticle may include apolymer-drug conjugate. For example, a drug may be conjugated to adisclosed polymer or copolymer (e.g., PLA-PEG), and such a polymer-drugconjugate may form part of a disclosed nanoparticle. For example, adisclosed therapeutic nanoparticle may optionally include from about 0.2to about 30 weight percent of a PLA-PEG or PLGA-PEG, wherein the PEG isfunctionalized with a drug (e.g., PLA-PEG-Drug).

In another example, a disclosed therapeutic nanoparticle may optionallyinclude from 0.2 to 30 weight percent of a PLA-PEG or PLGA-PEG, whereinthe PEG is functionalized with a drug (e.g., PLA-PEG-Drug).

A disclosed polymeric conjugate (e.g., a polymer-ligand conjugate) maybe formed using any suitable conjugation technique. For instance, twocompounds such as a targeting moiety or drug and a biocompatible polymer(e.g., a biocompatible polymer and a poly(ethylene glycol)) may beconjugated together using techniques such as EDC-NHS chemistry(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride andN-hydroxysuccinimide) or a reaction involving a maleimide or acarboxylic acid, which can be conjugated to one end of a thiol, anamine, or a similarly functionalized polyether. The conjugation of atargeting moiety or drug and a polymer to form a polymer-targetingmoiety conjugate or a polymer-drug conjugate can be performed in anorganic solvent, such as, but not limited to, dichloromethane,acetonitrile, chloroform, dimethylformamide, tetrahydrofuran, acetone,or the like. Specific reaction conditions can be determined by those ofordinary skill in the art using no more than routine experimentation.

In another set of embodiments, a conjugation reaction may be performedby reacting a polymer that comprises a carboxylic acid functional group(e.g., a poly(ester-ether) compound) with a polymer or other moiety(such as a targeting moiety or therapeutic drug) having an aminefunctionality thereon. For instance, a targeting moiety, such as alow-molecular weight ligand, or the therapeutic agent as may be reactedwith an amine to form an amine-containing moiety, which can then beconjugated to the carboxylic acid of the polymer. Such a reaction mayoccur as a single-step reaction, i.e., the conjugation is performedwithout using intermediates such as N-hydroxysuccinimide or a maleimide.In some embodiments, the therapeutic agent may be reacted with anamine-containing linker to form an amine-containing drug, which can thenbe conjugated to the carboxylic acid of the polymer as described above.The conjugation reaction between the amine-containing moiety and thecarboxylic acid-terminated polymer (such as a poly(ester-ether)compound) may be achieved, in one set of embodiments, by adding theamine-containing moiety, solubilized in an organic solvent such as (butnot limited to) dichloromethane, acetonitrile, chloroform,tetrahydrofuran, acetone, formamide, dimethylformamide, pyridines,dioxane, or dimethylsulfoxide, to a solution containing the carboxylicacid-terminated polymer. The carboxylic acid-terminated polymer may becontained within an organic solvent such as, but not limited to,dichloromethane, acetonitrile, chloroform, dimethylformamide,tetrahydrofuran, or acetone. Reaction between the amine-containingmoiety and the carboxylic acid-terminated polymer may occurspontaneously, in some cases. Unconjugated reactants may be washed awayafter such reactions, and the polymer may be precipitated in solventssuch as, for instance, ethyl ether, hexane, methanol, or ethanol. Incertain embodiments, a conjugate may be formed between analcohol-containing moiety and carboxylic acid functional group of apolymer, which can be achieved similarly as described above forconjugates of amines and carboxylic acids.

Preparation of Nanoparticles

Another aspect of this disclosure is directed to systems and methods ofmaking disclosed nanoparticles. In some embodiments, using two or moredifferent polymers (e.g., copolymers, e.g., block copolymers) indifferent ratios and producing particles from the polymers (e.g.,copolymers, e.g., block copolymers), properties of the particles becontrolled. For example, one polymer (e.g., copolymer, e.g., blockcopolymer) may include a low-molecular weight ligand, while anotherpolymer (e.g., copolymer, e.g., block copolymer) may be chosen for itsbiocompatibility and/or its ability to control immunogenicity of theresultant particle.

In some embodiments, a solvent used in a nanoparticle preparationprocess (e.g., a nanoprecipitation process or a nanoemulsion process asdiscussed below) may include a hydrophobic acid, which may conferadvantageous properties to the nanoparticles prepared using the process.As discussed above, in some cases, the hydrophobic acid may improve drugloading of disclosed nanoparticles. Furthermore, in some instances, thecontrolled release properties of disclosed nanoparticles may be improvedby the use of the hydrophobic acid. In some cases, the hydrophobic acidmay be included in, for example, an organic solution or an aqueoussolution used in the process. In one embodiment, the therapeutic agentis combined with an organic solution and the hydrophobic acid andoptionally one or more polymers. The hydrophobic acid concentration in asolution used to dissolve the therapeutic agent is discussed above andmay, for example, range from about 1 weight percent and about 30 weightpercent or from 1 weight percent and 30 weight percent, etc.

In one set of embodiments, the particles are formed by providing asolution comprising one or more polymers, and contacting the solutionwith a polymer nonsolvent to produce the particle. The solution may bemiscible or immiscible with the polymer nonsolvent. For example, awater-miscible liquid such as acetonitrile may contain the polymers, andparticles are formed as the acetonitrile is contacted with water, apolymer nonsolvent, e.g., by pouring the acetonitrile into the water ata controlled rate. The polymer contained within the solution, uponcontact with the polymer nonsolvent, may then precipitate to formparticles such as nanoparticles. Two liquids are said to be “immiscible”or not miscible, with each other when one is not soluble in the other toa level of at least 10% by weight at ambient temperature and pressure.Typically, an organic solution (e.g., dichloromethane, acetonitrile,chloroform, tetrahydrofuran, acetone, formamide, dimethylformamide,pyridines, dioxane, dimethylsulfoxide, etc.) and an aqueous liquid(e.g., water, or water containing dissolved salts or other species, cellor biological media, ethanol, etc.) are immiscible with respect to eachother. For example, the first solution may be poured into the secondsolution (at a suitable rate or speed). In some cases, particles such asnanoparticles may be formed as the first solution contacts theimmiscible second liquid, e.g., precipitation of the polymer uponcontact causes the polymer to form nanoparticles while the firstsolution is poured into the second liquid, and in some cases, forexample, when the rate of introduction is carefully controlled and keptat a relatively slow rate, nanoparticles may form. The control of suchparticle formation can be readily optimized by one of ordinary skill inthe art using only routine experimentation.

Properties such as surface functionality, surface charge, size, zeta (ζ)potential, hydrophobicity, ability to control immunogenicity, and thelike, may be highly controlled using a disclosed process. For instance,a library of particles may be synthesized, and screened to identify theparticles having a particular ratio of polymers that allows theparticles to have a specific density of moieties (e.g., low-molecularweight ligands) present on the surface of the particle. This allowsparticles having one or more specific properties to be prepared, forexample, a specific size and a specific surface density of moieties,without an undue degree of effort. Accordingly, certain embodiments aredirected to screening techniques using such libraries, as well as anyparticles identified using such libraries. In addition, identificationmay occur by any suitable method. For instance, the identification maybe direct or indirect, or proceed quantitatively or qualitatively.

In some embodiments, already-formed nanoparticles are functionalizedwith a targeting moiety using procedures analogous to those describedfor producing ligand-functionalized polymeric conjugates. For example, afirst copolymer (PLGA-PEG, poly(lactide-co-glycolide) and poly(ethyleneglycol)) is mixed with the protonatable nitrogen-containing therapeuticagent to form particles. The particles are then associated with alow-molecular weight ligand to form nanoparticles that can be used forthe treatment of cancer. The particles can be associated with varyingamounts of low-molecular weight ligands in order to control the ligandsurface density of the nanoparticle, thereby altering the therapeuticcharacteristics of the nanoparticle. Furthermore, for example, bycontrolling parameters such as molecular weight, the molecular weight ofPEG, and the nanoparticle surface charge, very precisely controlledparticles may be obtained.

In another embodiment, a nanoemulsion process is provided, such as theprocess represented in FIGS. 1, 2A, and 2B. For example, a thetherapeutic agent, a hydrophobic acid, a first polymer (for example, adiblock co-polymer such as PLA-PEG or PLGA-PEG, either of which may beoptionally bound to a ligand) and an optional second polymer (e.g.,(PL(G)A-PEG or PLA), may be combined with an organic solution to form afirst organic phase. Such first phase may include about 1 to about 50%weight solids, about 5 to about 50% weight solids, about 5 to about 40%weight solids, about 1 to about 15% weight solids, or about 10 to about30% weight solids. The first organic phase may be combined with a firstaqueous solution to form a second phase. The organic solution caninclude, for example, toluene, methyl ethyl ketone, acetonitrile,tetrahydrofuran, ethyl acetate, isopropyl alcohol, isopropyl acetate,dimethylformamide, methylene chloride, dichloromethane, chloroform,acetone, benzyl alcohol, Tween™ 80, Span 80, or the like, andcombinations thereof. In an embodiment, the organic phase may includebenzyl alcohol, ethyl acetate, and combinations thereof. The secondphase can range from about 0.1 and 50 weight %, or from about 1 and 50weight %, or from about 5 and 40 weight %, or from about 1 and 15 weight%, solids. The aqueous solution can be water, optionally in combinationwith one or more of sodium cholate, ethyl acetate, polyvinyl acetate andbenzyl alcohol. In some embodiments, the pH of the aqueous phase may beselected based on the pK_(a) of the protonoated basic therapeutic agentand/or the pK_(a) of the hydrophobic acid. For example, in certainembodiments, the therapeutic agent, when protonated, may have a firstpK_(a), the hydrophobic acid may have a second pK_(a), and the aqueousphase may have a pH equal to a pK_(a) unit between the first pK_(a) andthe second pK_(a). In a particular embodiment, the pH of the aqueousphase may be equal to a pK_(a) unit that is about equidistant betweenthe first pK_(a) and the second pK_(a).

In another embodiment, the first phase may include 1 to 50% weightsolids, 5 to 50% weight solids, 5 to 40% weight solids, 1 to 15% weightsolids, or 10 to 30% weight solids. In an embodiment, the second phasecan range from 0.1 and 50 weight %, or from 1 and 50 weight %, or from 5and 40 weight %, or from 1 and 15 weight %, solids. In a particularembodiment, the pH of the aqueous phase may be equal to a pK_(a) unitthat is equidistant between the first pK_(a) and the second pK_(a).

For example, the oil or organic phase may use a solvent that is onlypartially miscible with the nonsolvent (water). Therefore, when mixed ata low enough ratio and/or when using water pre-saturated with theorganic solvents, the oil phase remains liquid. The oil phase may beemulsified into an aqueous solution and, as liquid droplets, shearedinto nanoparticles using, for example, high energy dispersion systems,such as homogenizers or sonicators. The aqueous portion of the emulsion,otherwise known as the “water phase”, may be surfactant solutionconsisting of sodium cholate and pre-saturated with ethyl acetate andbenzyl alcohol. In some instances, the organic phase (e.g., firstorganic phase) may include the basic therapeutic agent. Additionally, incertain embodiments, the aqueous solution (e.g., first aqueous solution)may include the substantially hydrophobic acid. In other embodiments,both the basic therapeutic agent and the substantially hydrophobic acidmay be dissolved in the organic phase.

Emulsifying the second phase to form an emulsion phase may be performed,for example, in one or two emulsification steps. For example, a primaryemulsion may be prepared, and then emulsified to form a fine emulsion.The primary emulsion can be formed, for example, using simple mixing, ahigh pressure homogenizer, probe sonicator, stir bar, or a rotor statorhomogenizer. The primary emulsion may be formed into a fine emulsionthrough the use of e.g., probe sonicator or a high pressure homogenizer,e.g., by using 1, 2, 3, or more passes through a homogenizer. Forexample, when a high pressure homogenizer is used, the pressure used maybe about 30 to about 60 psi, about 40 to about 50 psi, about 1000 toabout 8000 psi, about 2000 to about 4000 psi, about 4000 to about 8000psi, or about 4000 to about 5000 psi, e.g., about 2000, 2500, 4000 or5000 psi.

In another example, when a high pressure homogenizer is used, thepressure used may be 30 to 60 psi, 40 to 50 psi, 1000 to 8000 psi, 2000to 4000 psi, 4000 to 8000 psi, or 4000 to 5000 psi, e.g., 2000, 2500,4000 or 5000 psi.

In some cases, fine emulsion conditions, which can be characterized by avery high surface to volume ratio of the droplets in the emulsion, canbe chosen to maximize the solubility of the therapeutic agent andhydrophobic acid and form the desired HIP. In certain embodiments, underfine emulsion conditions, equilibration of dissolved components canoccur very quickly, i.e., faster than solidification of thenanoparticles. Thus, selecting a HIP based on, e.g., the pK_(a),difference between the protonated form of the therapeutic agent and thehydrophobic acid, or adjusting other parameters such as the pH of thefine emulsion and/or the pH of the quench solution, can have asignificant impact on the drug loading and release properties of thenanoparticles by dictating, for example, the formation of a HIP in thenanoparticle as opposed to diffusion of the therapeutic agent and/orhydrophobic acid out of the nanoparticle.

In some embodiments, the therapeutic agent and the substantiallyhydrophobic acid may be combined in the second phase prior toemulsifying the second phase. In some instances, the therapeutic agentand the substantially hydrophobic acid may form a hydrophobic ion pairprior to emulsifying the second phase. In other embodiments, thetherapeutic agent and the substantially hydrophobic acid may form ahydrophobic ion pair during emulsification of the second phase. Forexample, the therapeutic agent and the substantially hydrophobic acidmay be combined in the second phase substantially concurrently withemulsifying the second phase, e.g., the therapeutic agent and thesubstantially hydrophobic acid may be dissolved in separate solutions(e.g., two substantially immiscible solutions), which are then combinedduring emulsification. In another example, the therapeutic agent and thesubstantially hydrophobic acid may be dissolved in separate misciblesolutions that are then fed into second phase during emulsification.

Either solvent evaporation or dilution may be needed to complete theextraction of the solvent and solidify the particles. For better controlover the kinetics of extraction and a more scalable process, a solventdilution via aqueous quench may be used. For example, the emulsion canbe diluted into cold water to a concentration sufficient to dissolve allof the organic solvent to form a quenched phase. In some embodiments,quenching may be performed at least partially at a temperature of about5° C. or less. For example, water used in the quenching may be at atemperature that is less that room temperature (e.g., about 0 to about10° C., or about 0 to about 5° C.). In certain embodiments, the quenchmay be chosen having a pH that is advantageous for quenching theemulsion phase, e.g., by improving the properties of the nanoparticles,such as the release profile, or improving a nanoparticle parameter, suchas the drug loading. The pH of the quench may be adjusted by acid orbase titration, for example, or by appropriate selection of a buffer. Insome embodiments, the pH of the quench may be selected based on thepK_(a) of the protonoated basic therapeutic agent and/or the pK_(a) ofthe hydrophobic acid. For example, in certain embodiments, the basictherapeutic agent, when protonated, may have a first pK_(a), thehydrophobic acid may have a second pK_(a), and the emulsion phase may bequenched with an aqueous solution having a pH equal to a pK_(a) unitbetween the first pK_(a) and the second pK_(a). In some embodiments, theresultant quenched phase may also have a pH equal to a pK_(a) unitbetween the first pK_(a) and the second pK_(a). In a particularembodiment, the pH may be equal to a pK_(a), unit that is aboutequidistant between the first pK_(a) and the second pK_(a).

In certain embodiments, HIP formation can occur during or afteremulsification, e.g., as a result of equilibrium conditions in the fineemulsion. Without wishing to be bound by any theory, it is believed thatorganic-soluble counter ions (i.e., the hydrophobic acid) can facilitatediffusion of the therapeutic agent into a nanoparticle of an emulsion asa result of HIP formation. Without wishing to be bound by any theory,the HIP may remain in the nanoparticle before solidification of thenanoparticle since the solubility of the HIP in the nanoparticle ishigher than the solubility of the HIP in the aqueous phase of theemulsion and/or in the quench. For example, by selecting a pH for thequench that is between the pK_(a) of the basic therapeutic agent and thepK_(a) of the hydrophobic acid, formation of ionized therapeutic agentand hydrophobic acid can be optimized. However, selecting a pH that istoo high may tend to cause the hydrophobic acid to diffuse out of thenanoparticle, whereas selecting a pH that is too low may tend to causethe therapeutic agent to diffuse out of the nanoparticle.

In some embodiments, the pH of an aqueous solution used in ananoparticle formulation process (e.g., including, but not limited to,the aqueous phase, the emulsion phase, the quench, and the quenchedphase) may be independently selected and may be range from about 1 toabout 3, in some embodiments from about 2 to about 4, in someembodiments, from about 3 to about 5, in some embodiments, from about 4to about 6, in some embodiments, from about 5 to about 7, in someembodiments, from about 6 to about 8, in some embodiments, from about 7to about 9, and in some embodiments, from about 8 to about 10. Incertain embodiments, the pH of an aqueous solution used in ananoparticle formulation process may range from about 3 to about 4, insome embodiments from about 4 to about 5, in some embodiments, fromabout 5 to about 6, in some embodiments from about 6 to about 7, in someembodiments from about 7 to about 8, and in some embodiments from about8 to about 9.

In some embodiments, the pH of an aqueous solution used in ananoparticle formulation process (e.g., including, but not limited to,the aqueous phase, the emulsion phase, the quench, and the quenchedphase) may be independently selected and may be range from 1 to 3, insome embodiments from 2 to 4, in some embodiments, from 3 to 5, in someembodiments, from 4 to 6, in some embodiments, from 5 to 7, in someembodiments, from 6 to 8, in some embodiments, from 7 to 9, and in someembodiments, from 8 to 10. In certain embodiments, the pH of an aqueoussolution used in a nanoparticle formulation process may range from 3 to4, in some embodiments from 4 to 5, in some embodiments, from 5 to 6, insome embodiments from 6 to 7, in some embodiments from 7 to 8, and insome embodiments from 8 to 9.

In some embodiments, not all of the therapeutic agent is encapsulated inthe particles at this stage, and a drug solubilizer is added to thequenched phase to form a solubilized phase. The drug solubilizer may befor example, polysorbate 80 (Tween™ 80), Tween™ 20, polyvinylpyrrolidone, cyclodextran, sodium dodecyl sulfate, sodium cholate,diethylnitrosamine, sodium acetate, urea, glycerin, propylene glycol,glycofurol, poly(ethylene)glycol, bris(polyoxyethyleneglycol)dodecylether, sodium benzoate, sodium salicylate, polyoxyethylene (100) stearylether, or combinations thereof. For example, Tween™ 80 may be added tothe quenched nanoparticle suspension to solubilize the free drug andprevent the formation of drug crystals. In some embodiments, a ratio ofdrug solubilizer to the protonatable nitrogen-containing therapeuticagent is about 200:1 to about 10:1, or in some embodiments about 100:1to about 10:1.

In some embodiments, a ratio of drug solubilizer to the protonatablenitrogen-containing therapeutic agent is 200:1 to 10:1, or in someembodiments 100:1 to 10:1.

The solubilized phase may be filtered to recover the nanoparticles. Forexample, ultrafiltration membranes may be used to concentrate thenanoparticle suspension and substantially eliminate organic solvent,free drug (i.e., unencapsulated therapeutic agent), drug solubilizer,and other processing aids (surfactants). Exemplary filtration may beperformed using a tangential flow filtration system. For example, byusing a membrane with a pore size suitable to retain nanoparticles whileallowing solutes, micelles, and organic solvent to pass, nanoparticlescan be selectively separated. Exemplary membranes with molecular weightcut-offs of ranging about 300 to about 500 kDa (˜from about 5 to about25 nm) may be used. Exemplary membranes with molecular weight cut-offsof ranging 300 to 500 kDa (˜from 5 to 25 nm) may be used.

Diafiltration may be performed using a constant volume approach, meaningthe diafiltrate (cold deionized water, e.g., about 0 to about 5° C., or0 to about 10° C.) may added to the feed suspension at the same rate asthe filtrate is removed from the suspension. In some embodiments,filtering may include a first filtering using a first temperature ofabout 0 to about 5° C., or 0 to about 10° C., and a second temperatureof about 20 to about 30° C., or 15 to about 35° C. In some embodiments,filtering may include processing about 1 to about 30, in some casesabout 1 to about 15, or in some cases 1 to about 6 diavolumes. Forexample, filtering may include processing about 1 to about 30, or insome cases about 1 to about 6 diavolumes, at about 0 to about 5° C., andprocessing at least one diavolume (e.g., about 1 to about 15, about 1 toabout 3, or about 1 to about 2 diavolumes) at about 20 to about 30° C.In some embodiments, filtering comprises processing different diavolumesat different distinct temperatures.

In some embodiments, filtering may include a first filtering using afirst temperature of 0 to 5° C., or 0 to 10° C., and a secondtemperature of 20 to 30° C., or 15 to 35° C. In some embodiments,filtering may include processing 1 to 30, in some cases 1 to 15, or insome cases 1 to 6 diavolumes. For example, filtering may includeprocessing 1 to 30, or in some cases 1 to 6 diavolumes, at 0 to 5° C.,and processing at least one diavolume (e.g., 1 to 15, 1 to 3, or 1 to 2diavolumes) at 20 to 30° C.

After purifying and concentrating the nanoparticle suspension, theparticles may be passed through one, two or more sterilizing and/ordepth filters, for example, using ˜0.2 μm depth pre-filter. For example,a sterile filtration step may involve filtering the therapeuticnanoparticles using a filtration train at a controlled rate. In someembodiments, the filtration train may include a depth filter and asterile filter.

In another embodiment of preparing nanoparticles, an organic phase isformed composed of a mixture of the therapeutic agent, and polymer(homopolymer, co-polymer, and co-polymer with ligand). The organic phaseis mixed with an aqueous phase at approximately a 1:5 ratio (oilphase:aqueous phase) where the aqueous phase is composed of a surfactantand some dissolved solvent. The primary emulsion is formed by thecombination of the two phases under simple mixing or through the use ofa rotor stator homogenizer. The primary emulsion is then formed into afine emulsion through the use of a high pressure homogenizer. The fineemulsion is then quenched by addition to deionized water under mixing.In some embodiments, the quench:emulsion ratio may be about 2:1 to about40:1, or in some embodiments about 5:1 to about 15:1. In someembodiments, the quench:emulsion ratio is approximately 8.5:1. In someembodiments, the quench:emulsion ratio may be 2:1 to 40:1, or in someembodiments 5:1 to 15:1. In some embodiments, the quench:emulsion ratiois 8.5:1. Then a solution of Tween™ (e.g., Tween™ 80) is added to thequench to achieve approximately 2% Tween™ overall. This serves todissolve free, unencapsulated therapeutic agent. The nanoparticles arethen isolated through either centrifugation orultrafiltration/diafiltration.

It will be appreciated that the amounts of polymer, therapeutic agent,and hydrophobic acid that are used in the preparation of the formulationmay differ from a final formulation. For example, some of thetherapeutic agent may not become completely incorporated in ananoparticle and such free therapeutic agent may be e.g., filtered away.For example, in an embodiment, a first organic solution containing about11 weight percent theoretical loading of therapeutic agent in a firstorganic solution containing about 9% of a first hydrophobic acid (e.g.,a fatty acid), a second organic solution containing about 89 weightpercent polymer (e.g., the polymer may include about 2.5 mol percent ofa targeting moiety conjugated to a polymer and about 97.5 mol percentPLA-PEG), and an aqueous solution containing about 0.12% of a secondhydrophobic acid (e.g., a bile acid) may be used in the preparation of aformulation that results in, e.g., a final nanoparticle comprising about2 weight percent therapeutic agent, about 97.5 weight percent polymer(where the polymer may include about 1.25 mol percent of a targetingmoiety conjugated to a polymer and about 98.75 mol percent PLA-PEG), andabout 0.5% total hydrophobic acid. Such processes may provide finalnanoparticles suitable for administration to a subject that includesabout 1 to about 20 percent by weight therapeutic agent, e.g., about 1,about 2, about 3, about 4, about 5, about 8, about 10, or about 15percent therapeutic agent by weight.

In another embodiment, a first organic solution containing 11 weightpercent theoretical loading of therapeutic agent in a first organicsolution containing 9% of a first hydrophobic acid (e.g., a fatty acid),a second organic solution containing 89 weight percent polymer (e.g.,the polymer may include 2.5 mol percent of a targeting moiety conjugatedto a polymer and 97.5 mol percent PLA-PEG), and an aqueous solutioncontaining 0.12% of a second hydrophobic acid (e.g., a bile acid) may beused in the preparation of a formulation that results in, e.g., a finalnanoparticle comprising 2 weight percent therapeutic agent, 97.5 weightpercent polymer (where the polymer may include 1.25 mol percent of atargeting moiety conjugated to a polymer and 98.75 mol percent PLA-PEG),and 0.5% total hydrophobic acid. Such processes may provide finalnanoparticles suitable for administration to a subject that includes 1to 20 percent by weight therapeutic agent, e.g., 1, 2, 3, 4, 5, 8, 10,or 15 percent therapeutic agent by weight.

In certain embodiments, the therapeutic nanoparticle comprises thetherapeutic agent1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand pamoic acid in a weight ratio of therapeutic agent to pamoic acid ofabout 0.1:1, about 0.5:1, about 1:1, about 1.1:1, about 1.2:1, about1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1,about 1.9:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1,about 4.5:1, about 5:1, about 5.5:1, about 6:1, about 6.5:1, about 7:1,about 7.5:1, about 8:1, about 8.5:1, about 9:1, about 9.5:1, or about10:1. In some embodiments, the therapeutic nanoparticle comprisesPLA-PEG (in a 16:5 molar ratio) in a weight ratio of therapeutic agentto PLA-PEG of about 0.5:1, about 1:1, about 1:2, about 1:3, about 1:4,about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about1:15, or about 1:20. In certain embodiments, the therapeuticnanoparticle comprises the therapeutic agent1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,pamoic acid in a weight ratio of therapeutic agent to pamoic acid ofabout 1.8:1, PLA-PEG (in a 16:5 molar ratio) in a weight ratio oftherapeutic agent to PLA-PEG of about 1:3, and PLA-PEG-GL in a weightratio of PLA-PEG to PLA-PEG-GL of about 44:1. In other embodiments, thetherapeutic nanoparticle additionally comprises a solubilizer. Incertain such embodiments, the solubilizer is polyoxyethylene (100)stearyl ether. In certain embodiments, the therapeutic nanoparticlecomprises the therapeutic agent1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand pamoic acid in a weight ratio of therapeutic agent to pamoic acid of0.1:1, 0.5:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1,1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1,6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, or 10:1. In some embodiments,the therapeutic nanoparticle comprises PLA-PEG (in a 16:5 molar ratio)in a weight ratio of therapeutic agent to PLA-PEG of 0.5:1, 1:1, 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, or 1:20. In certainembodiments, the therapeutic nanoparticle comprises the therapeuticagent1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,pamoic acid in a weight ratio of therapeutic agent to pamoic acid of1.8:1, PLA-PEG (in a 16:5 molar ratio) in a weight ratio of therapeuticagent to PLA-PEG of 1:3, and PLA-PEG-GL in a weight ratio of PLA-PEG toPLA-PEG-GL of 44:1. In other embodiments, the therapeutic nanoparticleadditionally comprises a solubilizer. In certain such embodiments, thesolubilizer is polyoxyethylene (100) stearyl ether.

In certain embodiments, the therapeutic nanoparticle comprises thetherapeutic agent1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand oleic acid in a weight ratio of therapeutic agent to oleic acid ofabout 0.1:1, about 0.5:1, about 1:1, about 1.1:1, about 1.2:1, about1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1,about 1.9:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1,about 4.5:1, about 5:1, about 5.5:1, about 6:1, about 6.5:1, about 7:1,about 7.5:1, about 8:1, about 8.5:1, about 9:1, about 9.5:1, or about10:1. In some embodiments, the therapeutic nanoparticle comprisesPLA-PEG (in a 16:5 molar ratio) in a weight ratio of therapeutic agentto PLA-PEG of about 0.5:1, about 1:1, about 1:2, about 1:3, about 1:4,about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:20, about1:25, or about 1:30. In certain embodiments, the therapeuticnanoparticle comprises the therapeutic agent1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,oleic acid in a weight ratio of therapeutic agent to oleic acid of about6:1, PLA-PEG (in a 16:5 molar ratio) in a weight ratio of therapeuticagent to PLA-PEG of about 1:7, and PLA-PEG-GL in a weight ratio ofPLA-PEG to PLA-PEG-GL of about 46:1. In some embodiments, thetherapeutic nanoparticle further comprises cholic acid. In otherembodiments, the therapeutic nanoparticle additionally comprises asolubilizer. In certain such embodiments, the solubilizer is polysorbate80. In certain embodiments, the therapeutic nanoparticle comprises thetherapeutic agent1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand oleic acid in a weight ratio of therapeutic agent to oleic acid of0.1:1, 0.5:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1,1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1,6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, or 10:1. In some embodiments,the therapeutic nanoparticle comprises PLA-PEG (in a 16:5 molar ratio)in a weight ratio of therapeutic agent to PLA-PEG of 0.5:1, 1:1, 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15,1:20, 1:25, or 1:30. In certain embodiments, the therapeuticnanoparticle comprises the therapeutic agent1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,oleic acid in a weight ratio of therapeutic agent to oleic acid of 6:1,PLA-PEG (in a 16:5 molar ratio) in a weight ratio of therapeutic agentto PLA-PEG of 1:7, and PLA-PEG-GL in a weight ratio of PLA-PEG toPLA-PEG-GL of 46:1. In some embodiments, the therapeutic nanoparticlefurther comprises cholic acid. In other embodiments, the therapeuticnanoparticle additionally comprises a solubilizer. In certain suchembodiments, the solubilizer is polysorbate 80.

In some embodiments, the therapeutic nanoparticle is a nanoparticleprepared by emulsification of a first organic phase comprising a firstpolymer, a therapeutic agent, and a substantially hydrophobic acid,thereby forming an emulsion phase; quenching of the emulsion phasethereby forming a quenched phase; and filtration of the quenched phaseto recover the therapeutic nanoparticles, wherein the therapeutic agentis1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor a pharmaceutically acceptable salt thereof.

In other embodiments, the therapeutic nanoparticle is a nanoparticleprepared by the process combining a first organic phase with a firstaqueous solution to form a second phase; emulsifying the second phase toform an emulsion phase, wherein the emulsion phase comprises a firstpolymer, therapeutic agent, and a substantially hydrophobic acid;quenching of the emulsion phase thereby forming a quenched phase; andfiltering the quenched phase to recover the therapeutic nanoparticles,wherein the therapeutic agent is1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,the first organic phase comprises the therapeutic agent and pamoic acidin a weight ratio of therapeutic agent to pamoic acid of about 11:1 andPLA-PEG (in a 16:5 molar ratio) in a weight ratio of therapeutic agentto PLA-PEG of about 1:3 in an organic solvent comprising of benzylalcohol and ethyl acetate in a weight ratio of benzyl alcohol to ethylacetate of about 1.25 and the first aqueous solution comprises apolyoxyethylene (100) stearyl ether dissolved in benzyl alcohol in aweight ratio of 0.005:1 and combining the first organic phase and thefirst aqueous phase in a weight ratio of about 1:5 to form a secondphase and emulsifying the second phase formed therefrom and quenchingthe emulsion phase with 0.1 M citric acid in water solution at pH 4.5and concentrating the resulting product.

The therapeutic agent may include alternative forms such aspharmaceutically acceptable salt forms, free base forms, hydrates,isomers, and prodrugs thereof.

An “effective amount” when used in connection with a compound of thisinvention is an amount effective for inhibiting mTOR or PI3K in asubject.

The therapeutic agent of the present invention exhibits an mTORinhibitory activity and therefore, the therapeutic nanoparticle preparedfrom the therapeutic agent can be utilized to inhibit abnormal cellgrowth in which mTOR plays a role. Thus, the therapeutic nanoparticle ofthe present invention is effective in the treatment of disorders withwhich abnormal cell growth actions of mTOR are associated, such asrestenosis, atherosclerosis, bone disorders, arthritis, diabeticretinopathy, psoriasis, benign prostatic hypertrophy, atherosclerosis,inflammation, angiogenesis, immunological disorders, pancreatitis,kidney disease, cancer, etc. In particular, the compounds of the presentinvention possess excellent cancer cell growth inhibiting effects andare effective in treating cancers, preferably all types of solid cancersand malignant lymphomas, and especially, leukemia, skin cancer, bladdercancer, breast cancer, uterus cancer, ovary cancer, prostate cancer,lung cancer, colon cancer, pancreas cancer, renal cancer, gastriccancer, brain tumor, advanced renal cell carcinoma, acute lymphoblasticleukemia, malignant melanoma, soft-tissue or bone sarcoma, etc.

The therapeutic agent of the present invention exhibits a PI3 kinaseinhibitory activity and, therefore, the therapeutic nanoparticleprepared from the therapeutic agent can be utilized to inhibit abnormalcell growth in which PI3 kinases play a role. Thus, the therapeuticagent of the present invention are effective in the treatment ofdisorders with which abnormal cell growth actions of PI3 kinases areassociated, such as restenosis, atherosclerosis, bone disorders,arthritis, diabetic retinopathy, psoriasis, benign prostatichypertrophy, atherosclerosis, inflammation, angiogenesis, immunologicaldisorders, pancreatitis, kidney disease, cancer, etc. In particular, thetherapeutic nanoparticle of the present invention possess excellentcancer cell growth inhibiting effects and are effective in treatingcancers, preferably all types of solid cancers and malignant lymphomas,and especially, leukemia, skin cancer, bladder cancer, breast cancer,uterus cancer, ovary cancer, prostate cancer, lung cancer, colon cancer,pancreas cancer, renal cancer, gastric cancer, brain tumor, head andneck cancer e.g., cancer of the following regions: Oral cavity, Pharynx,Larynx, Paranasal sinuses and nasal cavity, or Salivary glands),advanced renal cell carcinoma, acute lymphoblastic leukemia, malignantmelanoma, soft-tissue or bone sarcoma, etc.

The therapeutic agent is also useful in treating a cancer associatedwith PTEN deficiency. Phosphatase and tensin homolog deleted onchromosome 10 (PTEN) is a lipid and protein phosphatase and functions asa protein phosphatase by dephosphorylating protein substrates on serine,threonine, and tyrosine residues. PTEN also functions as a lipidphosphatase by dephosphorylating phophoinosital 3,4,5-triphosphate(PIP3), a key signaling component of the phosphoinositol-3-kinase(PI3-kinase). PTEN is a known tumor suppressor that has been implicatedin cellular processes including mediation of the MAP kinase signalingpathway, centromeric maintenance, and is implicated in DNA repairpathways through mediation of Rad51 gene expression. Tumor suppressorsplay roles in maintaining genome stability, and loss of function ofthese tumor suppressors is known to result in genomic instability.Genetic instability represents an inevitable consequence of the loss oftumor suppressors. Indeed, the frequent occurrence of PTEN mutation andgenetic instability is found in a large range of PTEN-deficient cancers.Likewise, it is known that several tumor cell lines are PTEN deficient.Likewise, it is known that several tumor cell lines are PTEN deficient.PTEN-null embryonic stem cells were shown to exhibit DNA repaircheckpoint defects in response to ionizing radiation, which results inthe accumulation of unrepaired chromosomes with DNA double-strand gapsand breaks. Further mechanistic study revealed that the observed G2checkpoint defects may result from functional impairment of thecheckpoint protein, CHK1, due to lack of PTEN. PTEN deficiency directlyelevates AKT kinase activity, which triggers CHK1 phosphorylation.Phosphorylated CHK1 undergoes ubiquitination, which prevents its entryinto the nucleus. Sequestering CHK1 in the cytoplasm impairs its normalfunction in initiating a DNA repair checkpoint. In addition, CHK1inactivation in PTEN-deficient cells leads to the accumulation of DNAdouble-strand breaks. Examination of CHK1 localization in a large panelof primary human breast carcinomas indicates an increased cytoplasmiclevel of CHK1 in tumor cells with lower expression of PTEN and elevatedAKT phosphorylation. Furthermore, aneuploidy was frequently observed inboth human breast carcinomas with low expression of PTEN and prostaticintraepithelial neoplasia from Pten.sup.+/−mice. Such in vitro and invivo observations indicate that PTEN deficiencies are involved ininitiation of an oncogenic signaling process by causing dysfunction ofimportant checkpoint proteins. The cytoplasm has been considered as theprimary site for PTEN to elicit its tumor-suppressive function, and theability of PTEN to block the PI3-kinase pathway through its phosphataseactivity has been regarded as the key mechanism by which PTEN suppressescarcinogenesis. Although the cellular distribution of PTEN varies indifferent tissues, endogenous PTEN in neurons, gliomas and cells of thethyroid, pancreas and skin is found mostly in the nuclear compartment.Growing evidence indicate that malignancies may be accompanied bytranslocation of PTEN from the nucleus to the cytoplasm. Inactivation ofPTEN, either by mutations, deletions, or promoter hypermethylation, hasbeen identified in a wide variety of tumors. The therapeutic agent ofthe present invention a method of treating a cancer associated with aPTEN deficiency such as endometrial carcinoma, glioblastoma(glioblastoma multiforme/anaplastic astrocytoma), prostate cancer, renalcancer, small cell lung carcinoma, meningioma, head and neck cancer,thyroid cancer, bladder cancer, colorectal cancer, breast cancer,melanoma.

Pharmaceutical Formulations

Nanoparticles disclosed herein may be combined with pharmaceuticallyacceptable carriers to form a pharmaceutical composition, according toanother aspect. As would be appreciated by one of skill in this art, thecarriers may be chosen based on the route of administration as describedbelow, the location of the target issue, the drug being delivered, thetime course of delivery of the drug, etc.

The pharmaceutical compositions can be administered to a patient orsubject by any means known in the art including oral and parenteralroutes. The term “patient” or “subject” as used herein areinterchangeable and refer to humans as well as non-humans, including,for example, mammals, birds, reptiles, amphibians, and fish. Forinstance, the non-humans may be mammals (e.g., a rodent, a mouse, a rat,a rabbit, a monkey, a dog, a cat, a primate, or a pig). In certainembodiments parenteral routes are desirable since they avoid contactwith the digestive enzymes that are found in the alimentary canal.According to such embodiments, inventive compositions may beadministered by injection (e.g., intravenous, subcutaneous orintramuscular, intraperitoneal injection), rectally, vaginally,topically (as by powders, creams, ointments, or drops), or by inhalation(as by sprays).

In a particular embodiment, the nanoparticles are administered to asubject in need thereof systemically, e.g., by IV infusion or injection.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P., and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Inone embodiment, the inventive conjugate is suspended in a carrier fluidcomprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) Tween™80. The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, theencapsulated or unencapsulated conjugate is mixed with at least oneinert, pharmaceutically acceptable excipient or carrier such as sodiumcitrate or dicalcium phosphate and/or (a) fillers or extenders such asstarches, lactose, sucrose, glucose, mannitol, and silicic acid, (b)binders such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectantssuch as glycerol, (d) disintegrating agents such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate, (e) solution retarding agents such as paraffin,(f) absorption accelerators such as quaternary ammonium compounds, (g)wetting agents such as, for example, cetyl alcohol and glycerolmonostearate, (h) absorbents such as kaolin and bentonite clay, and (i)lubricants such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof. Inthe case of capsules, tablets, and pills, the dosage form may alsocomprise buffering agents.

It will be appreciated that the exact dosage of a nanoparticlecontaining the therapeutic agent is chosen by the individual physicianin view of the patient to be treated, in general, dosage andadministration are adjusted to provide an effective amount of thetherapeutic agent nanoparticle to the patient being treated. As usedherein, the “effective amount” of a nanoparticle containing aprotonatable nitrogen-containing therapeutic agent refers to the amountnecessary to elicit the desired biological response. As will beappreciated by those of ordinary skill in this art, the effective amountof a nanoparticle containing the therapeutic agent may vary depending onsuch factors as the desired biological endpoint, the drug to bedelivered, the target tissue, the route of administration, etc. Forexample, the effective amount of a nanoparticle containing thetherapeutic agent might be the amount that results in a reduction intumor size by a desired amount over a desired period of time. Additionalfactors which may be taken into account include the severity of thedisease state; age, weight and gender of the patient being treated;diet, time and frequency of administration; drug combinations; reactionsensitivities; and tolerance/response to therapy.

The nanoparticles may be formulated in dosage unit form for ease ofadministration and uniformity of dosage. The expression “dosage unitform” as used herein refers to a physically discrete unit ofnanoparticle appropriate for the patient to be treated. It will beunderstood, however, that the total daily usage of the compositions willbe decided by the attending physician within the scope of sound medicaljudgment. For any nanoparticle, the therapeutically effective dose canbe estimated initially either in cell culture assays or in animalmodels, usually mice, rabbits, dogs, or pigs. The animal model is alsoused to achieve a desirable concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans. Therapeutic efficacy andtoxicity of nanoparticles can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED₅₀ (thedose is therapeutically effective in 50% of the population) and LD₅₀(the dose is lethal to 50% of the population). The dose ratio of toxicto therapeutic effects is the therapeutic index, and it can be expressedas the ratio, LD₅₀/ED₅₀. Pharmaceutical compositions which exhibit largetherapeutic indices may be useful in some embodiments. The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosage for human use.

In an embodiment, compositions disclosed herein may include about 10 ppmof palladium or less, about 8 ppm of palladium or less, or about 6 ppmof palladium or less. For example, provided here is a composition thatincludes nanoparticles having a polymeric conjugate wherein thecomposition has less than about 10 ppm of palladium or less.

In an embodiment, compositions disclosed herein may include 10 ppm ofpalladium or less, 8 ppm of palladium or less, or 6 ppm of palladium orless. For example, provided here is a composition that includesnanoparticles having a polymeric conjugate wherein the composition hasless than 10 ppm of palladium or less.

In some embodiments, a composition suitable for freezing iscontemplated, including nanoparticles disclosed herein and a solutionsuitable for freezing, e.g., a sugar such as a mono, di, or polysaccharide, e.g., sucrose and/or a trehalose, and/or a salt and/or acyclodextrin solution is added to the nanoparticle suspension. The sugar(e.g., sucrose or trehalose) may act, e.g., as a cryoprotectant toprevent the particles from aggregating upon freezing. For example,provided herein is a nanoparticle formulation comprising a plurality ofdisclosed nanoparticles, sucrose, an ionic halide, and water; whereinthe nanoparticles/sucrose/water/ionic halide is about3-40%/10-40%/20-95%/0.1-10% (w/w/w/w) or about 5-10%/10-15%/80-90%/1-10%(w/w/w/w). For example, such solution may include nanoparticles asdisclosed herein, about 5% to about 20% by weight sucrose and an ionichalide such as sodium chloride, in a concentration of about 10-100 mM.In another example, provided herein is a nanoparticle formulationcomprising a plurality of disclosed nanoparticles, trehalose,cyclodextrin, and water, wherein thenanoparticles/trehalose/water/cyclodextrin is about3-40%/1-25%/20-95%/1-25% (w/w/w/w) or about 5-10%/1-25%/80-90%/10-15%(w/w/w/w).

In another example, provided herein is a nanoparticle formulationcomprising a plurality of disclosed nanoparticles, sucrose, an ionichalide, and water, wherein the nanoparticles/sucrose/water/ionic halideis 3-40%/10-40%/20-95%/0.1-10% (w/w/w/w) or 5-10%/10-15%/80-90%/1-10%(w/w/w/w). For example, such solution may include nanoparticles asdisclosed herein, 5% to 20% by weight sucrose and an ionic halide suchas sodium chloride, in a concentration of 10-100 mM. In another example,provided herein is a nanoparticle formulation comprising a plurality ofdisclosed nanoparticles, trehalose, cyclodextrin, and water; wherein thenanoparticles/trehalose/water/cyclodextrin is 3-40%/1-25%/20-95%/1-25%(w/w/w/w) or 5-10%/1-25%/80-90%/10-15% (w/w/w/w).

For example, a contemplated solution may include nanoparticles asdisclosed herein, about 1% to about 25% by weight of a disaccharide suchas trehalose or sucrose (e.g., about 5% to about 25% trehalose orsucrose, e.g. about 10% trehalose or sucrose, or about 15% trehalose orsucrose, e.g. about 5% sucrose) by weight) and a cyclodextrin such asβ-cyclodextrin, in a concentration of about 1% to about 25% by weight(e.g. about 5% to about 20%, e.g. 10% or about 20% by weight, or about15% to about 20% by weight cyclodextrin). Contemplated formulations mayinclude a plurality of disclosed nanoparticles (e.g. nanoparticleshaving PLA-PEG and an active agent), and about 2% to about 15 wt %/o (orabout 4% to about 6 wt %, e.g. about 5 wt %) sucrose and about 5 wt % toabout 20% (e.g. about 7% wt percent to about 12 wt %/o, e.g. about 10 wt%) of a cyclodextrin, e.g., HPbCD).

In another example, a contemplated solution may include nanoparticles asdisclosed herein, 1% to 25% by weight of a disaccharide such astrehalose or sucrose (e.g., 5% to 25% trehalose or sucrose, e.g. 10%trehalose or sucrose, or 15% trehalose or sucrose, e.g. 5% sucrose) byweight) and a cyclodextrin such as β-cyclodextrin, in a concentration of1% to 25% by weight (e.g. 5% to 20%, e.g. 10% or 20% by weight, or 15%to 20% by weight cyclodextrin). Contemplated formulations may include aplurality of disclosed nanoparticles (e.g. nanoparticles having PLA-PEGand an active agent), and 2% to 15 wt % (or 4% to 6 wt %, e.g. 5 wt %)sucrose and 5 wt % to 20% (e.g. 7% wt percent to 12 wt %, e.g. 10 wt %)of a cyclodextrin, e.g., HPbCD).

The present disclosure relates in part to lyophilized pharmaceuticalcompositions that, when reconstituted, have a minimal amount of largeaggregates. Such large aggregates may have a size of about 0.5 μm orgreater, about 1 μm or greater, about 10 μm or greater, and can beundesirable in a reconstituted solution. Aggregate sizes can be measuredusing a variety of techniques including those indicated in the U.S.Pharmacopeia (“USP”) at <788>, hereby incorporated by reference. Thetests outlined in USP <788> include a light obscuration particle counttest, microscopic particle count test, laser diffraction, and singleparticle optical sensing. In one embodiment, the particle size in agiven sample is measured using laser diffraction and/or single particleoptical sensing.

The USP <788> by light obscuration particle count test sets forthguidelines for sampling particle sizes in a suspension. For solutionswith less than or equal to 100 mL, the preparation complies with thetest if the average number of particles present does not exceed 6000 percontainer that are ≧10 μm and 600 per container that are ≧25 μm.

As outlined in USP <788>, the microscopic particle count test sets forthguidelines for determining particle amounts using a binocular microscopeadjusted to 100±10× magnification having an ocular micrometer. An ocularmicrometer is a circular diameter graticule that consists of a circledivided into quadrants with black reference circles denoting 10 μm and25 μm when viewed at 100× magnification. A linear scale is providedbelow the graticule. The number of particles with reference to 10 μm and25 μm are visually tallied. For solutions with less than or equal to 100mL, the preparation complies with the test if the average number ofparticles present does not exceed 3000 per container that are ≧10 μm and300 per container that are ≧25 μm.

In some embodiments, a 10 mL aqueous sample of a disclosed compositionupon reconstitution comprises less than 600 particles per mL having asize greater than or equal to 10 microns; and/or less than 60 particlesper mL having a size greater than or equal to 25 microns.

Dynamic light scattering (DLS) may be used to measure particle size, butit relies on Brownian motion so the technique may not detect some largerparticles. Laser diffraction relies on differences in the index ofrefraction between the particle and the suspension media. The techniqueis capable of detecting particles at the sub-micron to millimeter range.Relatively small (e.g., about 1-5 weight %) amounts of larger particlescan be determined in nanoparticle suspensions. Single particle opticalsensing (SPOS) uses light obscuration of dilute suspensions to countindividual particles of about 0.5 μm. By knowing the particleconcentration of the measured sample, the weight percentage ofaggregates or the aggregate concentration (particles/mL) can becalculated.

Formation of aggregates can occur during lyophilization due to thedehydration of the surface of the particles. This dehydration can beavoided by using lyoprotectants, such as disaccharides, in thesuspension before lyophilization. Suitable disaccharides includesucrose, lactulose, lactose, maltose, trehalose, or cellobiose, and/ormixtures thereof. Other contemplated disaccharides include kojibiose,nigerose, isomaltose, β,β-trehalose, α,β-trehalose, sophorose,laminaribiose, gentiobiose, turanose, maltulose, palatinose,gentiobiulose, mannobiase, melibiose, melibiulose, rutinose, rutinulose,and xylobiose. Reconstitution shows equivalent DLS size distributionswhen compared to the starting suspension. However, laser diffraction candetect particles of >10 μm in size in some reconstituted solutions.Further, SPOS also may detect >10 μm sized particles at a concentrationabove that of the FDA guidelines (10⁴-10⁵ particles/mL for >10 μmparticles).

In some embodiments, one or more ionic halide salts may be used as anadditional lyoprotectant to a sugar, such as sucrose, trehalose ormixtures thereof. Sugars may include disaccharides, monosaccharides,trisaccharides, and/or polysaccharides, and may include otherexcipients, e.g. glycerol and/or surfactants. Optionally, a cyclodextrinmay be included as an additional lyoprotectant. The cyclodextrin may beadded in place of the ionic halide salt. Alternatively, the cyclodextrinmay be added in addition to the ionic halide salt.

Suitable ionic halide salts may include sodium chloride, calciumchloride, zinc chloride, or mixtures thereof. Additional suitable ionichalide salts include potassium chloride, magnesium chloride, ammoniumchloride, sodium bromide, calcium bromide, zinc bromide, potassiumbromide, magnesium bromide, ammonium bromide, sodium iodide, calciumiodide, zinc iodide, potassium iodide, magnesium iodide, or ammoniumiodide, and/or mixtures thereof. In one embodiment, about 1 to about 15weight percent sucrose may be used with an ionic halide salt. In oneembodiment, 1 to 15 weight percent sucrose may be used with an ionichalide salt. In one embodiment, the lyophilized pharmaceuticalcomposition may comprise about 10 to about 100 mM sodium chloride. Inone embodiment, the lyophilized pharmaceutical composition may comprise10 to 100 mM sodium chloride. In another embodiment, the lyophilizedpharmaceutical composition may comprise about 100 to about 500 mM ofdivalent ionic chloride salt, such as calcium chloride or zinc chloride.In another embodiment, the lyophilized pharmaceutical composition maycomprise 100 to 500 mM of divalent ionic chloride salt, such as calciumchloride or zinc chloride. In yet another embodiment, the suspension tobe lyophilized may further comprise a cyclodextrin, for example, about 1to about 25 weight percent of cyclodextrin may be used. In yet anotherembodiment, the suspension to be lyophilized may further comprise acyclodextrin, for example, 1 to 25 weight percent of cyclodextrin may beused.

A suitable cyclodextrin may include α-cyclodextrin, β-cyclodextrin,γ-cyclodextrin, or mixtures thereof. Exemplary cyclodextrinscontemplated for use in the compositions disclosed herein includehydroxypropyl-β-cyclodextrin (HPbCD), hydroxyethyl-β-cyclodextrin,sulfobutylether-β-cyclodextrin, methyl-β-cyclodextrin,dimethyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin, carboxymethylethyl-β-cyclodextrin, diethyl-β-cyclodextrin,tri-O-alkyl-β-cyclodextrin, glocosyl-β-cyclodextrin, andmaltosyl-β-cyclodextrin. In one embodiment, about 1 to about 25 weightpercent trehalose (e.g. about 10% to about 15%, e.g. 5 to about 20% byweight) may be used with cyclodextrin. In one embodiment, thelyophilized pharmaceutical composition may comprise about 1 to about 25weight percent 3-cyclodextrin. An exemplary composition may comprisenanoparticles comprising PLA-PEG, an active/therapeutic agent, about 4%to about 6% (e.g. about 5% wt percent) sucrose, and about 8 to about 12weight percent (e.g. about 10 wt percent) HPbCD. In one embodiment, 1 to25 weight percent trehalose (e.g. 10% to 15%, e.g. 5 to 20% by weight)may be used with cyclodextrin. In one embodiment, the lyophilizedpharmaceutical composition may comprise 1 to 25 weight percentβ-cyclodextrin. An exemplary composition may comprise nanoparticlescomprising PLA-PEG, an active/therapeutic agent, 4% to 6% (e.g. 5%0/wtpercent) sucrose, and 8 to 12 weight percent (e.g. 10 wt percent) HPbCD.

In one aspect, a lyophilized pharmaceutical composition is providedcomprising disclosed nanoparticles, wherein upon reconstitution of thelyophilized pharmaceutical composition at a nanoparticle concentrationof about 50 mg/mL, in less than or about 100 mL of an aqueous medium,the reconstituted composition suitable for parenteral administrationcomprises less than 6000, such as less than 3000, microparticles ofgreater than or equal to 10 microns; and/or less than 600, such as lessthan 300, microparticles of greater than or equal to 25 microns.

The number of microparticles can be determined by means known to one ofordinary skill in the art, such as described in USP <788>; by lightobscuration particle count test, such as described in USP <788>; bymicroscopic particle count test, laser diffraction, and single particleoptical sensing.

In an aspect, a pharmaceutical composition suitable for parenteral useupon reconstitution is provided comprising a plurality of therapeuticparticles each comprising a copolymer having a hydrophobic polymersegment and a hydrophilic polymer segment; an active agent; a sugar; anda cyclodextrin.

For example, the copolymer may be poly(lactic)acid-block-poly(ethylene)glycol copolymer. Upon reconstitution, a 100 mLaqueous sample may comprise less than 6000 particles having a sizegreater than or equal to 10 microns; and less than 600 particles havinga size greater than or equal to 25 microns.

The step of adding a disaccharide and an ionic halide salt may compriseadding about 5 to about 15 weight percent sucrose or about 5 to about 20weight percent trehalose (e.g., about 10 to about 20 weight percenttrehalose), and about 10 to about 500 mM ionic halide salt. The ionichalide salt may be selected from sodium chloride, calcium chloride, andzinc chloride, or mixtures thereof. In an embodiment, about 1 to about25 weight percent cyclodextrin is also added.

In another embodiment, the step of adding a disaccharide and an ionichalide salt may comprise adding 5 to 15 weight percent sucrose or 5 to20 weight percent trehalose (e.g., 10 to 20 weight percent trehalose),and 10 to 500 mM ionic halide salt. In an embodiment, 1 to 25 weightpercent cyclodextrin is also added.

In another embodiment, the step of adding a disaccharide and acyclodextrin may comprise adding about 5 to about 15 weight percentsucrose or about 5 to about 20 weight percent trehalose (e.g., about 10to about 20 weight percent trehalose), and about 1 to about 25 weightpercent cyclodextrin. In an embodiment, about 10 to about 15 weightpercent cyclodextrin is added. The cyclodextrin may be selected fromα-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or mixtures thereof.

In another embodiment, the step of adding a disaccharide and acyclodextrin may comprise adding 5 to 15 weight percent sucrose or 5 to20 weight percent trehalose (e.g., 10 to 20 weight percent trehalose),and 1 to 25 weight percent cyclodextrin. In an embodiment, 10 to 15weight percent cyclodextrin is added.

In another aspect, a method of preventing substantial aggregation ofparticles in a pharmaceutical nanoparticle composition is providedcomprising adding a sugar and a salt to the lyophilized formulation toprevent aggregation of the nanoparticles upon reconstitution. In anembodiment, a cyclodextrin is also added to the lyophilized formulation.In yet another aspect, a method of preventing substantial aggregation ofparticles in a pharmaceutical nanoparticle composition is providedcomprising adding a sugar and a cyclodextrin to the lyophilizedformulation to prevent aggregation of the nanoparticles uponreconstitution.

A contemplated lyophilized composition may have a therapeutic particleconcentration of greater than about 40 mg/mL. The formulation suitablefor parenteral administration may have less than about 600 particleshaving a size greater than 10 microns in a 10 mL dose. Lyophilizing maycomprise freezing the composition at a temperature of greater than about−40° C., or e.g. less than about −30° C., forming a frozen composition;and drying the frozen composition to form the lyophilized composition.The step of drying may occur at about 50 mTorr at a temperature of about−25 to about −34° C., or about −30 to about −34° C.

A contemplated lyophilized composition may have a therapeutic particleconcentration of greater than 40 mg/mL. The formulation suitable forparenteral administration may have less than 600 particles having a sizegreater than 10 microns in a 10 mL dose. Lyophilizing may comprisefreezing the composition at a temperature of greater than −40° C., ore.g. less than −30° C., forming a frozen composition; and drying thefrozen composition to form the lyophilized composition. The step ofdrying may occur at 50 mTorr at a temperature of −25 to −34° C., or −30to −34° C.

Methods of Treatment

In some embodiments, targeted nanoparticles may be used for the purposeof treatment. As used herein the terms “treat”, “treatment”, or“treating” mean to alleviate, ameliorate, relieve, delay onset of,inhibit progression of, reduce severity of, and/or reduce incidence ofone or more symptoms or features of a disease, disorder, and/orcondition. In some embodiments, targeted nanoparticles may be used totreat solid tumors, e.g., cancer and/or cancer cells. In certainembodiments, targeted nanoparticles or pharmaceutical compositionscomprising the nanoparticles may be used to treat any cancer whereinprostate-specific membrane antigen (PSMA) is expressed on the surface ofcancer cells or in the tumor neovasculature in a subject in needthereof, including the neovasculature of prostate or non-prostate solidtumors. Examples of the PSMA-related indication include, but are notlimited to, prostate cancer, breast cancer, non-small cell lung cancer,colorectal carcinoma, and glioblastoma.

In some embodiments, targeted nanoparticles or pharmaceuticalcompositions comprising the nanoparticles may be used for thepreparation of a medicament to alleviate, ameliorate, relieve, delayonset of, inhibit progression of, reduce severity of, and/or reduceincidence of one or more symptoms or features of a disease, disorder,and/or condition. In some embodiments, targeted nanoparticles orpharmaceutical compositions comprising the nanoparticles may be used forthe preparation of a medicament to treat any cancer whereinprostate-specific membrane antigen (PSMA) is expressed on the surface ofcancer cells or in the tumor neovasculature in a subject in needthereof, including the neovasculature of prostate or non-prostate solidtumors.

The term “cancer” includes pre-malignant as well as malignant cancers.Cancers include, but are not limited to, blood (e.g., chronicmyelogenous leukemia, chronic myelomonocytic leukemia, Philadelphiachromosome positive acute lymphoblastic leukemia, mantle cell lymphoma),prostate, gastric cancer, colorectal cancer, skin cancer, e.g.,melanomas or basal cell carcinomas, lung cancer (e.g., non-small celllung cancer), breast cancer, cancers of the head and neck, bronchuscancer, pancreatic cancer, urinary bladder cancer, brain or centralnervous system cancer, peripheral nervous system cancer, esophagealcancer, cancer of the oral cavity or pharynx, liver cancer (e.g.,hepatocellular carcinoma), kidney cancer (e.g., renal cell carcinoma),testicular cancer, biliary tract cancer, small bowel or appendix cancer,gastrointestinal stromal tumor, salivary gland cancer, thyroid glandcancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer ofhematological tissues head or neck cancer, and the like. “Cancer cells”can be in the form of a tumor (i.e., a solid tumor), exist alone withina subject (e.g., leukemia cells), or be cell lines derived from acancer.

Cancer can be associated with a variety of physical symptoms. Symptomsof cancer generally depend on the type and location of the tumor. Forexample, lung cancer can cause coughing, shortness of breath, and chestpain, while colon cancer often causes diarrhea, constipation, and bloodin the stool. However, to give but a few examples, the followingsymptoms are often generally associated with many cancers: fever,chills, night sweats, cough, dyspnea, weight loss, loss of appetite,anorexia, nausea, vomiting, diarrhea, anemia, jaundice, hepatomegaly,hemoptysis, fatigue, malaise, cognitive dysfunction, depression,hormonal disturbances, neutropenia, pain, non-healing sores, enlargedlymph nodes, peripheral neuropathy, and sexual dysfunction.

In one aspect, a method for the treatment of cancer (e.g., leukemia) isprovided. In some embodiments, the treatment of cancer comprisesadministering a therapeutically effective amount of inventive targetedparticles to a subject in need thereof, in such amounts and for suchtime as is necessary to achieve the desired result. In certainembodiments, a “therapeutically effective amount” of an inventivetargeted particle is that amount effective for treating, alleviating,ameliorating, relieving, delaying onset of, inhibiting progression of,reducing severity of, and/or reducing incidence of one or more symptomsor features of cancer.

In one aspect, a method for administering inventive compositions to asubject suffering from cancer (e.g., leukemia) is provided. In someembodiments, particles may be administered to a subject in such amountsand for such time as is necessary to achieve the desired result (i.e.,treatment of cancer). In certain embodiments, a “therapeuticallyeffective amount” of an inventive targeted particle is that amounteffective for treating, alleviating, ameliorating, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of cancer.

Inventive therapeutic protocols involve administering a therapeuticallyeffective amount of an inventive targeted particle to a healthyindividual (i.e., a subject who does not display any symptoms of cancerand/or who has not been diagnosed with cancer). For example, healthyindividuals may be “immunized” with an inventive targeted particle priorto development of cancer and/or onset of symptoms of cancer; at riskindividuals (e.g., patients who have a family history of cancer;patients carrying one or more genetic mutations associated withdevelopment of cancer; patients having a genetic polymorphism associatedwith development of cancer, patients infected by a virus associated withdevelopment of cancer, patients with habits and/or lifestyles associatedwith development of cancer, etc.) can be treated substantiallycontemporaneously with (e.g., within 48 hours, within 24 hours, orwithin 12 hours of) the onset of symptoms of cancer. Of courseindividuals known to have cancer may receive inventive treatment at anytime.

In other embodiments, disclosed nanoparticles can be used to inhibit thegrowth of cancer cells, e.g., myelogenous leukemia cancer cells. As usedherein, the term “inhibits growth of cancer cells” or “inhibiting growthof cancer cells” refers to any slowing of the rate of cancer cellproliferation and/or migration, arrest of cancer cell proliferationand/or migration, or killing of cancer cells, such that the rate ofcancer cell growth is reduced in comparison with the observed orpredicted rate of growth of an untreated control cancer cell. The term“inhibits growth” can also refer to a reduction in size or disappearanceof a cancer cell or tumor, as well as to a reduction in its metastaticpotential. Preferably, such an inhibition at the cellular level mayreduce the size, deter the growth, reduce the aggressiveness, or preventor inhibit metastasis of a cancer in a patient. Those skilled in the artcan readily determine, by any of a variety of suitable indicia, whethercancer cell growth is inhibited.

Inhibition of cancer cell growth may be evidenced, for example, byarrest of cancer cells in a particular phase of the cell cycle, e.g.,arrest at the G2/M phase of the cell cycle. Inhibition of cancer cellgrowth can also be evidenced by direct or indirect measurement of cancercell or tumor size. In human cancer patients, such measurementsgenerally are made using well known imaging methods such as magneticresonance imaging, computerized axial tomography and X-rays. Cancer cellgrowth can also be determined indirectly, such as by determining thelevels of circulating carcinoembryonic antigen, prostate specificantigen or other cancer-specific antigens that are correlated withcancer cell growth. Inhibition of cancer growth is also generallycorrelated with prolonged survival and/or increased health andwell-being of the subject.

Also provided herein are methods of administering to a patient ananoparticle disclosed herein including an active agent, wherein, uponadministration to a patient, such nanoparticles substantially reducesthe volume of distribution and/or substantially reduces free C_(max), ascompared to administration of the agent alone (i.e., not as a disclosednanoparticle).

In some embodiments, the therapeutic nanoparticle is administered with acompound selected from the group consisting of a topoisomerase Iinhibitor, a MEK 1/2 inhibitor, a HSP90 inhibitor, procarbazine,dacarbazine, gemcitabine, capecitabine, methotrexate, taxol, taxotere,mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide,ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin,dacarbazine, procarbizine, etoposide, teniposide, campathecins,bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin,plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epirubicin,5-fluorouracil, docetaxel, paclitaxel, leucovorin, levamisole,irinotecan, estramustine, etoposide, nitrogen mustards, BCNU,carmustine, lomustine, vinblastine, vincristine, vinorelbine,oxaliplatin, imatinib mesylate, bevacizumab, hexamethylmelamine,topotecan, tyrosine kinase inhibitors, tyrphostins, herbimycin A,genistein, erbstatin, hydroxyzine, glatiramer acetate, interferonbeta-1a, interferon beta-1b, natalizumab and lavendustin A; and apharmaceutically acceptable carrier.

In another aspect, there is provided a therapeutic nanoparticle asdescribed herein for use as a medicament in a subject.

In yet another aspect, there is provided a therapeutic nanoparticle asdescribed herein for use in the production of an anti-proliferativeeffect in a subject.

In still another aspect, there is provided a therapeutic nanoparticle asdescribed herein for use in a subject as an anti-invasive agent in thecontainment and/or treatment of solid tumor disease.

In yet another aspect, there is provided the use of a therapeuticnanoparticle as described herein in the prevention or treatment ofcancer in a subject.

In still another aspect, there is provided a therapeutic nanoparticle asdescribed herein for use in the prevention or treatment of cancer in asubject.

In yet another aspect, there is provided the use of a therapeuticnanoparticle as described herein in the manufacture of a medicament forthe prevention or treatment of cancer in a subject.

In still another aspect, there is provided the use of a therapeuticnanoparticle as described herein for the production of ananti-proliferative effect in a subject.

In yet another aspect, there is provided the use of a therapeuticnanoparticle as described herein in the manufacture of a medicament foruse in the production of an anti-proliferative effect in a subject.

In still another aspect, there is provided the use of a therapeuticnanoparticle as described herein in the manufacture of a medicament foruse in a subject as an anti-invasive agent in the containment and/ortreatment of solid tumor disease.

In yet another aspect, there is provided a method for producing ananti-proliferative effect in a subject in need of such treatment whichcomprises administering to said subject an effective amount of atherapeutic nanoparticle as described herein.

In still another aspect, there is provided a method for producing ananti-invasive effect by the containment and/or treatment of solid tumordisease in a subject in need of such treatment which comprisesadministering to said subject an effective amount of a therapeuticnanoparticle as described herein.

In yet another aspect, there is provided a therapeutic nanoparticle asdescribed herein for use in the prevention or treatment of solid tumordisease in a subject.

In still another aspect, there is provided the use of a therapeuticnanoparticle as described herein in the manufacture of a medicament foruse in the prevention or treatment of solid tumor disease in a subject.

In yet another aspect, there is provided a method for the prevention ortreatment of solid tumor disease in a subject in need of such treatmentwhich comprises administering to said subject an effective amount of atherapeutic nanoparticle as described herein.

U.S. Pat. No. 8,206,747, issued Jun. 26, 2012, entitled “Drug LoadedPolymeric Nanoparticles and Methods of Making and Using Same” is herebyincorporated by reference in its entirety.

EMBODIMENTS

Some embodiments of this invention are as follows:

1. A therapeutic nanoparticle comprising:

about 0.05 to about 30 weight percent of a substantially hydrophobicacid;

about 0.2 to about 25 weight percent of a therapeutic agent; wherein thepK_(a) of the protonated therapeutic agent is at least about 1.0 pK_(a)units greater than the pK_(a) of the hydrophobic acid; and

about 50 to about 99.75 weight percent of a polymer selected fromdiblock poly(lactic) acid-poly(ethylene)glycol copolymer or a diblockpoly(lactic acid-co-glycolic acid)-poly(ethylene)glycol copolymer andcombination, wherein the therapeutic nanoparticle comprises about 10 toabout 30 weight percent poly(ethylene)glycol, wherein the therapeuticagent is1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor a pharmaceutically acceptable salt thereof.

2. The therapeutic nanoparticle of embodiment 1 wherein the amount ofthe therapeutic agent is about 0.2 to about 20 weight percent.

3. The therapeutic nanoparticle according to embodiment 1 or 2comprising:

-   1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea;-   and PLA-PEG (in a 16:5 molar ratio) in a weight ratio of about 1:7    1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea:PLA-PEG.    4. The therapeutic nanoparticle according to embodiment 1 or 2    comprising:-   1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea;-   and PLA-PEG (in a 16:5 molar ratio) in a weight ratio of about 1:14    1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea:PLA-PEG.    5. The therapeutic nanoparticle according to embodiment 1 or 2    comprising:-   1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea;-   and PLA-PEG (in a 16:5 molar ratio) in a weight ratio of about 1:5    1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea:PLA-PEG.    6. A therapeutic nanoparticle comprising:

about 0.2 to about 25 weight percent of a therapeutic agent;

a substantially hydrophobic acid, wherein the molar ratio of thesubstantially hydrophobic acid to the therapeutic agent ranges fromabout 0.25:1 to about 2:1 and wherein the pK_(a) of the protonatedtherapeutic agent is at least about 1.0 pK_(a) units greater than thepK_(a) of the hydrophobic acid; and

about 50 to about 99.75 weight percent of a polymer selected fromdiblock poly(lactic) acid-poly(ethylene)glycol copolymer or a diblockpoly(lactic acid-co-glycolic acid)-poly(ethylene)glycol copolymer andcombination, wherein the therapeutic nanoparticle comprises about 10 toabout 30 weight percent poly(ethylene)glycol, wherein the therapeuticagent is1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor a pharmaceutically acceptable salt thereof.

7. The therapeutic nanoparticle of embodiment 6, wherein the amount ofthe therapeutic agent is about 0.2 to about 20 weight percent.

8. A therapeutic nanoparticle comprising:

a substantially hydrophobic acid;

a therapeutic agent; wherein the pK_(a) of the protonated therapeuticagent is at least about 1.0 pK_(a) units greater than the pK_(a) of thehydrophobic acid; and

a polymer selected from diblock poly(lactic) acid-poly(ethylene)glycolcopolymer or a diblock poly(lactic acid-co-glycolicacid)-poly(ethylene)glycol copolymer and combination thereof, whereinthe therapeutic agent is1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor a pharmaceutically acceptable salt thereof.

9. A therapeutic nanoparticle comprising:

a therapeutic agent;

a substantially hydrophobic acid, wherein the molar ratio of thesubstantially hydrophobic acid to the therapeutic agent ranges fromabout 0.25:1 to about 2:1 and wherein the pK_(a) of the protonatedtherapeutic agent is at least about 1.0 pK_(a) units greater than thepK_(a) of the hydrophobic acid; and

a polymer selected from diblock poly(lactic) acid-poly(ethylene)glycolcopolymer or a diblock poly(lactic acid-co-glycolicacid)-poly(ethylene)glycol copolymer and combination thereof, and,wherein the therapeutic agent is1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor a pharmaceutically acceptable salt thereof.

10. The therapeutic nanoparticle of embodiment 6 or 9, wherein the molarratio of the substantially hydrophobic acid to the therapeutic agent isabout 0.5:1 to about 1.5:1.

11. The therapeutic nanoparticle of embodiment 6 or 9, wherein the molarratio of the substantially hydrophobic acid to the therapeutic agent isabout 0.25:1 to about 1:1.

12. The therapeutic nanoparticle of embodiment 6 or 9, wherein the molarratio of the substantially hydrophobic acid to the therapeutic agent isabout 0.75:1 to about 1.25:1.

13. The therapeutic nanoparticle of any one of embodiments 1-12, whereinthe pK_(a) of the protonated therapeutic agent is at least about 2.0pK_(a) units greater than the pK_(a) of the hydrophobic acid.

14. The therapeutic nanoparticle of any one of embodiments 1-12, whereinthe pK_(a) of the protonated therapeutic agent is at least about 4.0pK_(a) units greater than the pK_(a) of the hydrophobic acid.

15. A therapeutic nanoparticle comprising:

a hydrophobic ion-pair comprising a hydrophobic acid and therapeuticagent; wherein the difference between the pK_(a) of the protonatedtherapeutic agent and the hydrophobic acid is at least about 1.0 pK_(a)units or greater; and

about 50 to about 99.75 weight percent of a diblock poly(lactic)acid-poly(ethylene)glycol copolymer, wherein the poly(lactic)acid-poly(ethylene)glycol copolymer has a number average molecularweight of about 15 kDa to about 20 kDa poly(lactic acid) and a numberaverage molecular weight of about 4 kDa to about 6 kDapoly(ethylene)glycol, wherein the therapeutic agent is1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor a pharmaceutically acceptable salt thereof.

16. The therapeutic nanoparticle of embodiment 15, wherein thedifference between the pK_(a) of the protonated therapeutic agent andthe hydrophobic acid is at least about 2.0 pK_(a) units.

17. The therapeutic nanoparticle of embodiment 15, wherein thedifference between the pK_(a) of the protonated therapeutic agent andthe hydrophobic acid is at least about 4.0 pK_(a) units.

18. The therapeutic nanoparticle of any one of embodiments 1-5, 8 or13-17, comprising about 0.05 to about 20 weight percent of thehydrophobic acid.

19. The therapeutic nanoparticle of any one of embodiments 1-18, whereinthe substantially hydrophobic acid has a log P ranging from about 2 toabout 7.

20. The therapeutic nanoparticle of any one of embodiments 1-18, whereinthe substantially hydrophobic acid has a log P ranging from about 4 toabout 8.

21. The therapeutic nanoparticle of any one of embodiments 1-20, whereinthe substantially hydrophobic acid has a pK_(a) in water from about −1.0to about 5.0.

22. The therapeutic nanoparticle of any one of embodiments 1-20, whereinthe substantially hydrophobic acid has a pK_(a) in water from about 2.0to about 5.0.

23. The therapeutic nanoparticle of any one of embodiments 1-22, whereinthe substantially hydrophobic acid and the therapeutic agent form ahydrophobic ion pair in the therapeutic nanoparticle.

24. The therapeutic nanoparticle of any one of embodiments 1-23, whereinthe hydrophobic acid is a fatty acid.

25. The therapeutic nanoparticle of embodiment 24, wherein the fattyacid is a saturated fatty acid selected from the group consisting of:caproic acid, enanthic acid, caprylic acid, pelargonic acid, capricacid, undecanoic acid, lauric acid, tridecylic acid, myristic acid,pentadecylic acid, palmitic acid, margaric acid, stearic acid,nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid,tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid,heptacosylic acid, montanic acid, nonacosylic acid, melissic acid,henatriacontylic acid, lacceroic acid, psyllic acid, geddic acid,ceroplastic acid, hexatriacontylic acid, and combinations thereof.26. The therapeutic nanoparticle of embodiment 24, wherein the fattyacid is an omega-3 fatty acid selected from the group consisting of:hexadecatrienoic acid, alpha-linolenic acid, stearidonic acid,eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid,heneicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid,tetracosapentaenoic acid, tetracosahexaenoic acid, and combinationsthereof.27. The therapeutic nanoparticle of embodiment 24, wherein the fattyacid is an omega-6 fatty acid selected from the group consisting of:linoleic acid, gamma-linolenic acid, eicosadienoic acid,dihomo-gamma-linolenic acid, arachidonic acid, docosadienoic acid,adrenic acid, docosapentaenoic acid, tetracosatetraenoic acid,tetracosapentaenoic acid, and combinations thereof.28. The therapeutic nanoparticle of embodiment 24, wherein the fattyacid is an omega-9 fatty acid selected from the group consisting of:oleic acid, eicosenoic acid, mead acid, erucic acid, nervonic acid, andcombinations thereof.29. The therapeutic nanoparticle of embodiment 28, wherein the fattyacid is oleic acid.30. The therapeutic nanoparticle of embodiment 29 wherein the weightratio of1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureato oleic acid is about 6:1.31. The therapeutic nanoparticle of embodiment 24, wherein the fattyacid is a polyunsaturated fatty acid selected from the group consistingof: rumenic acid, α-calendic acid, β-calendic acid, jacaric acid,α-eleostearic acid, β-eleostearic acid, catalpic acid, punicic acid,rumelenic acid, α-parinaric acid, β-parinaric acid, bosseopentaenoicacid, pinolenic acid, podocarpic acid, and combinations thereof.32. The therapeutic nanoparticle of any one of embodiments 1-24, whereinthe hydrophobic acid is a bile acid.33. The therapeutic nanoparticle of embodiment 32, wherein the bile acidis selected from the group consisting of chenodeoxycholic acid,ursodeoxycholic acid, deoxycholic acid, hycholic acid, beta-muricholicacid, cholic acid, lithocholic acid, an amino acid-conjugated bile acid,and combinations thereof.34. The therapeutic nanoparticle of embodiment 33, wherein the bile acidis cholic acid.35. The therapeutic nanoparticle of embodiment 33, wherein the aminoacid-conjugated bile acid is a glycine-conjugated bile acid or ataurine-conjugated bile acid.36. The therapeutic nanoparticle of any one of embodiments 1-23, whereinthe hydrophobic acid is selected from the group consisting of dioctylsulfosuccinic acid, 1-hydroxy-2-naphthoic acid, dodecylsulfuric acid,naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, pamoicacid, undecanoic acid, and combinations thereof.37. The therapeutic nanoparticle of embodiment 36, wherein thehydrophobic acid is pamoic acid.38. The therapeutic nanoparticle of embodiment 37 wherein the weightratio of1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureato pamoic acid is about 1.8:1.39. The therapeutic nanoparticle of any one of embodiments 1-38,comprising about 1 to about 20 weight percent of the therapeutic agent.40. The therapeutic nanoparticle of any one of embodiments 1-38,comprising about 2 to about 20 weight percent of the therapeutic agent.41. The therapeutic nanoparticle of any one of embodiments 1-38,comprising about 4 to about 20 weight percent of the therapeutic agent.42. The therapeutic nanoparticle of any one of embodiments 1-38,comprising about 5 to about 20 weight percent of the therapeutic agent.43. The therapeutic nanoparticle of any one of embodiments 1-38, whereinthe hydrophobic acid has a molecular weight of between about 200 Da andabout 800 Da.44. The therapeutic nanoparticle of any one of embodiments 1-43, whereinthe therapeutic nanoparticle substantially retains the therapeutic agentfor at least 1 minute when placed in a phosphate buffer solution at 37°C.45. The therapeutic nanoparticle of any one of embodiments 1-43, whereinthe therapeutic nanoparticle substantially immediately releases lessthan about 30% of the therapeutic agent when placed in a phosphatebuffer solution at 37° C.46. The therapeutic nanoparticle of any one of embodiments 1-43, whereinthe therapeutic nanoparticle releases about 10 to about 45% of thetherapeutic agent over about 1 hour when placed in a phosphate buffersolution at 37° C.47. The therapeutic nanoparticle of any one of embodiments 1-43, whereinthe therapeutic nanoparticle releases about 0.01 to about 15% of thetherapeutic agent over about 4 hours when placed in a phosphate buffersolution at 37° C.48. The therapeutic nanoparticle of any one of embodiments 1-43, whereinthe therapeutic nanoparticle releases about 0.01 to about 15% of thetherapeutic agent over about 10 hours when placed in a phosphate buffersolution at 37° C.49. The therapeutic nanoparticle of any one of embodiments 1-43, whereinthe therapeutic nanoparticle releases about 0.01 to about 25% of thetherapeutic agent over about 20 hours when placed in a phosphate buffersolution at 37° C.50. The therapeutic nanoparticle of any one of embodiments 1-43, whereinthe therapeutic nanoparticle releases about 1 to about 40% of thetherapeutic agent over about 40 hours when placed in a phosphate buffersolution at 37° C.51. The therapeutic nanoparticle of any one of embodiments 1-43, whereinthe therapeutic nanoparticle has a release profile that is substantiallythe same as a release profile for a control nanoparticle that issubstantially the same as the therapeutic nanoparticle except that itdoes not contain a fatty acid or bile acid.52. The therapeutic nanoparticle of any one of embodiments 1-51, whereinthe poly(lactic) acid-poly(ethylene)glycol copolymer has a poly(lactic)acid number average molecular weight fraction of about 0.6 to about0.95.53. The therapeutic nanoparticle of any one of embodiments 1-51, whereinthe poly(lactic) acid-poly(ethylene)glycol copolymer has a poly(lactic)acid number average molecular weight fraction of about 0.6 to about 0.8.54. The therapeutic nanoparticle of any one of embodiments 1-51, whereinthe poly(lactic) acid-poly(ethylene)glycol copolymer has a poly(lactic)acid number average molecular weight fraction of about 0.75 to about0.85.55. The therapeutic nanoparticle of any one of embodiments 1-51, whereinthe poly(lactic) acid-poly(ethylene)glycol copolymer has a poly(lactic)acid number average molecular weight fraction of about 0.7 to about 0.9.56. The therapeutic nanoparticle of any one of embodiments 1-55, whereinthe therapeutic nanoparticle comprises about 10 to about 25 weightpercent poly(ethylene)glycol.57. The therapeutic nanoparticle of any one of embodiments 1-55, whereinthe therapeutic nanoparticle comprises about 10 to about 20 weightpercent poly(ethylene)glycol.58. The therapeutic nanoparticle of any one of embodiments 1-55, whereinthe therapeutic nanoparticle comprises about 15 to about 25 weightpercent poly(ethylene)glycol.59. The therapeutic nanoparticle of any one of embodiments 1-55, whereinthe therapeutic nanoparticle comprises about 20 to about 30 weightpercent poly(ethylene)glycol.60. The therapeutic nanoparticle of any one of embodiments 1-59, whereinthe poly(lactic) acid-poly(ethylene)glycol copolymer has a numberaverage molecular weight of about 15 kDa to about 20 kDa poly(lacticacid) and a number average molecular weight of about 4 kDa to about 6kDa poly(ethylene)glycol.61. The therapeutic nanoparticle of any one of embodiments 1-60, furthercomprising about 0.2 to about 30 weight percent poly(lactic)acid-poly(ethylene)glycol copolymer functionalized with a targetingligand.62. The therapeutic nanoparticle of any one of embodiments 1-60, furthercomprising about 0.2 to about 30 weight percent poly(lactic)acid-co-poly(glycolic) acid-poly(ethylene)glycol copolymerfunctionalized with a targeting ligand.63. The therapeutic nanoparticle of embodiment 61 or 62, wherein thetargeting ligand is covalently bound to the poly(ethylene)glycol.64. The therapeutic nanoparticle of any one of embodiments 1-63, whereinthe hydrophobic acid is a polyelectrolyte.65. The therapeutic nanoparticle of embodiment 64, wherein thepolyelectrolyte is selected from the group consisting of a poly(styrenesulfonic acid), polypolyacrylic acid, and polymethacrylic acid.66. The therapeutic nanoparticle of any one of embodiments 1-65, whereinthe substantially hydrophobic acid is a mixture of two or moresubstantially hydrophobic acids.67. The therapeutic nanoparticle of embodiment 66, comprising a mixtureof two substantially hydrophobic acids.68. The therapeutic nanoparticle of embodiment 67, wherein the twosubstantially hydrophobic acids are oleic acid and cholic acid.69. The therapeutic nanoparticle of embodiment 66, comprising a mixtureof three substantially hydrophobic acids.70. The therapeutic nanoparticle of embodiment 66, comprising a mixtureof four substantially hydrophobic acids.71. The therapeutic nanoparticle of embodiment 66, comprising a mixtureof five substantially hydrophobic acids.72. A therapeutic nanoparticle prepared by a process comprising thesteps of:

emulsification of a first organic phase comprising a first polymer, atherapeutic agent, and a substantially hydrophobic acid, thereby formingan emulsion phase;

quenching of the emulsion phase thereby forming a quenched phase; and

filtration of the quenched phase to recover the therapeuticnanoparticles, wherein the therapeutic agent is1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor a pharmaceutically acceptable salt thereof.

73. The therapeutic nanoparticle of embodiment 72, wherein thehydrophobic acid is a fatty acid.

74. The therapeutic nanoparticle of embodiment 73, wherein the fattyacid is a saturated fatty acid selected from the group consisting of:caproic acid, enanthic acid, caprylic acid, pelargonic acid, capricacid, undecanoic acid, lauric acid, tridecylic acid, myristic acid,pentadecylic acid, palmitic acid, margaric acid, stearic acid,nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid,tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid,heptacosylic acid, montanic acid, nonacosylic acid, melissic acid,henatriacontylic acid, lacceroic acid, psyllic acid, geddic acid,ceroplastic acid, hexatriacontylic acid, and combinations thereof.75. The therapeutic nanoparticle of embodiment 73, wherein the fattyacid is an omega-3 fatty acid selected from the group consisting of:hexadecatrienoic acid, alpha-linolenic acid, stearidonic acid,cicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid,heneicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid,tetracosapentaenoic acid, tetracosahexaenoic acid, and combinationsthereof.76. The therapeutic nanoparticle of embodiment 73, wherein the fattyacid is an omega-6 fatty acid selected from the group consisting of:linoleic acid, gamma-linolenic acid, eicosadienoic acid,dihomo-gamma-linolenic acid, arachidonic acid, docosadienoic acid,adrenic acid, docosapentaenoic acid, tetracosatetraenoic acid,tetracosapentaenoic acid, and combinations thereof.77. The therapeutic nanoparticle of embodiment 73, wherein the fattyacid is an omega-9 fatty acid selected from the group consisting of:oleic acid, eicosenoic acid, mead acid, erucic acid, nervonic acid, andcombinations thereof.78. The therapeutic nanoparticle of embodiment 77, wherein the fattyacid is oleic acid.79. The therapeutic nanoparticle of embodiment 73, wherein the fattyacid is a polyunsaturated fatty acid selected from the group consistingof: rumenic acid, α-calendic acid, β-calendic acid, jacaric acid,α-eleostearic acid, β-eleostearic acid, catalpic acid, punicic acid,rumelenic acid, α-parinaric acid, β-parinaric acid, bosseopentaenoicacid, pinolenic acid, podocarpic acid, and combinations thereof.80. The therapeutic nanoparticle of any one of embodiments 72, whereinthe hydrophobic acid is a bile acid.81. The therapeutic nanoparticle of embodiment 80, wherein the bile acidis selected from the group consisting of chenodeoxycholic acid,ursodeoxycholic acid, deoxycholic acid, hycholic acid, beta-muricholicacid, cholic acid, lithocholic acid, an amino acid-conjugated bile acid,and combinations thereof.82. The therapeutic nanoparticle of embodiment 81, wherein the bile acidis cholic acid.83. The therapeutic nanoparticle of embodiment 81, wherein the aminoacid-conjugated bile acid is a glycine-conjugated bile acid or ataurine-conjugated bile acid.84. The therapeutic nanoparticle of any one of embodiments 72, whereinthe hydrophobic acid is selected from the group consisting of dioctylsulfosuccinic acid, 1-hydroxy-2-naphthoic acid, dodecylsulfuric acid,naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, pamoicacid, undecanoic acid, and combinations thereof.85. The therapeutic nanoparticle of embodiment 84, wherein thehydrophobic acid is pamoic acid.86. The therapeutic nanoparticle of any one of embodiments 72-85,wherein the hydrophobic acid has a molecular weight of between about 200Da and about 800 Da.87. The therapeutic nanoparticle of any one of embodiments 72-86,wherein the therapeutic nanoparticle substantially retains thetherapeutic agent for at least 1 minute when placed in a phosphatebuffer solution at 37° C.88. The therapeutic nanoparticle of any one of embodiments 72-86,wherein the therapeutic nanoparticle substantially immediately releasesless than about 30% of the therapeutic agent when placed in a phosphatebuffer solution at 37° C.89. The therapeutic nanoparticle of any one of embodiments 72-86,wherein the therapeutic nanoparticle releases about 10 to about 45% ofthe therapeutic agent over about 1 hour when placed in a phosphatebuffer solution at 37° C.90. The therapeutic nanoparticle of any one of embodiments 72-86,wherein the therapeutic nanoparticle releases about 0.01 to about 15% ofthe therapeutic agent over about 4 hours when placed in a phosphatebuffer solution at 37° C.91. The therapeutic nanoparticle of any one of embodiments 72-86,wherein the therapeutic nanoparticle releases about 0.01 to about 15% ofthe therapeutic agent over about 10 hours when placed in a phosphatebuffer solution at 37° C.92. The therapeutic nanoparticle of any one of embodiments 72-86,wherein the therapeutic nanoparticle releases about 0.01 to about 25% ofthe therapeutic agent over about 20 hours when placed in a phosphatebuffer solution at 37° C.93. The therapeutic nanoparticle of any one of embodiments 72-86,wherein the therapeutic nanoparticle releases about 1 to about 40% ofthe therapeutic agent over about 40 hours when placed in a phosphatebuffer solution at 37° C.94. The therapeutic nanoparticle of any one of embodiments 72-86,wherein the therapeutic nanoparticle has a release profile that issubstantially the same as a release profile for a control nanoparticlethat is substantially the same as the therapeutic nanoparticle exceptthat it does not contain a fatty acid or bile acid.95. The therapeutic nanoparticle of any one of embodiments 72-94,wherein the first polymer is poly(lactic) acid-poly(ethylene)glycolcopolymer.96. The therapeutic nanoparticle of any one of embodiments 72-94,wherein the first polymer is poly(lactic) acid-co-poly(glycolic)acid-poly(ethylene)glycol copolymer.97. The therapeutic nanoparticle of any one of embodiments 72-96,wherein the substantially hydrophobic acid is a mixture of two or moresubstantially hydrophobic acids.98. The therapeutic nanoparticle of embodiment 97, comprising a mixtureof two substantially hydrophobic acids.99. The therapeutic nanoparticle of embodiment 97, comprising a mixtureof three substantially hydrophobic acids.100. The therapeutic nanoparticle of embodiment 97, comprising a mixtureof four substantially hydrophobic acids.101. The therapeutic nanoparticle of embodiment 97, comprising a mixtureof five substantially hydrophobic acids.102. The therapeutic nanoparticle of any of embodiments 1, 5-95, or97-101 wherein the polymer is PLA-PEG and the mole ratio of PLA-PEG is5:1.103. A therapeutic nanoparticle prepared by the process comprising thesteps of:

combining a first organic phase with a first aqueous solution to form asecond phase;

emulsifying the second phase to form an emulsion phase, wherein theemulsion phase comprises a first polymer, therapeutic agent, and asubstantially hydrophobic acid;

quenching of the emulsion phase thereby forming a quenched phase; and

filtering the quenched phase to recover the therapeutic nanoparticles,wherein the therapeutic agent is1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,the first organic phase comprises the therapeutic agent and pamoic acidin a weight ratio of therapeutic agent to pamoic acid of about 11:1 andPLA-PEG (in a 16:5 molar ratio) in a weight ratio of therapeutic agentto PLA-PEG of about 1:3 in an organic solvent comprising of benzylalcohol and ethyl acetate in a weight ratio of benzyl alcohol to ethylacetate of about 1.25 and the first aqueous solution comprises apolyoxyethylene (100) stearyl ether dissolved in benzyl alcohol in aweight ratio of 0.005:1 and combining the first organic phase and thefirst aqueous phase in a weight ratio of about 1:5 to form a secondphase and emulsifying the second phase formed therefrom and quenchingthe emulsion phase with 0.1 M citric acid in water solution at pH 4.5and concentrating the resulting product.

104. A therapeutic nanoparticle of1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor pharmaceutically acceptable salt thereof.

105. A therapeutic nanoparticle comprising of a therapeutic agent or apharmaceutically acceptable salt thereof and a polymer selected fromdiblock poly(lactic) acid-poly(ethylene)glycol copolymer or a diblockpoly(lactic acid-co-glycolic acid)-poly(ethylene)glycol copolymer andcombination thereof, wherein the therapeutic agent is1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor a pharmaceutically acceptable salt thereof.106. The therapeutic nanoparticle of any of embodiments 1-71, 104, or105, wherein a targeting ligand is additionally present and isPLA-PEG-GL, wherein GL has the following structure:

107. The therapeutic nanoparticle according to any one of embodiments1-71 or 104-106, further comprising a solubilizer.108. The therapeutic nanoparticle according to embodiment 107, whereinthe solubilizer is polysorbate 80.109. The therapeutic nanoparticle of embodiment 107, wherein thesolubilizer is polyoxyethylene (100) stearyl ether.110. The therapeutic nanoparticle of any one of embodiments 1-109,wherein the therapeutic agent is1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea.111. A pharmaceutical composition comprising a therapeutic nanoparticleof any of embodiments 1-110 and a pharmaceutically acceptable excipient.112. The pharmaceutical composition of embodiment 111 comprising aplurality of therapeutic nanoparticles.113. The pharmaceutical composition of embodiment 111 or 112, furthercomprising a saccharide.114. The pharmaceutical composition of any one of embodiments 111-113,further comprising a cyclodextrin.115. The pharmaceutical composition of embodiments 113 or 114, whereinthe saccharide is a disaccharide selected from the group consisting ofsucrose, trehalose, and a mixture thereof.116. A method of treating cancer in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of a therapeutic nanoparticle of any one of embodiments 1-110 ora pharmaceutical composition of any one of embodiments 111-115.117. The method of embodiment 116, wherein the cancer is chronicmyelogenous leukemia.118. The method of embodiment 116, wherein the cancer isgastrointestinal stromal tumor.119. The method of embodiment 116, wherein the cancer is selected fromthe group consisting of chronic myelomonocytic leukemia,hypereosinophilic syndrome, renal cell carcinoma, hepatocellularcarcinoma, Philadelphia chromosome positive acute lymphoblasticleukemia, non-small cell lung cancer, pancreatic cancer, breast cancer,a solid tumor, head and neck cancer and mantle cell lymphoma.120. The method of embodiment 119, wherein the cancer is breast cancer.121. A process for preparing a therapeutic nanoparticle, comprising thesteps of:

combining a first organic phase with a first aqueous solution to form asecond phase;

emulsifying the second phase to form an emulsion phase, wherein theemulsion phase comprises a first polymer, therapeutic agent, and asubstantially hydrophobic acid;

quenching of the emulsion phase thereby forming a quenched phase; and

filtering the quenched phase to recover the therapeutic nanoparticles,wherein the therapeutic agent is1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor a pharmaceutically acceptable salt thereof.

122. The process of embodiment 121, further comprising combining thetherapeutic agent and the substantially hydrophobic acid in the secondphase prior to emulsifying the second phase.

123. The process of embodiment 122, wherein the therapeutic agent andthe substantially hydrophobic acid form a hydrophobic ion pair prior toemulsifying the second phase.

124. The process of embodiment 122, wherein the therapeutic agent andthe substantially hydrophobic acid form a hydrophobic ion pair priorduring emulsification of the second phase.

125. The process of embodiment 121, further comprising combining thetherapeutic agent and the substantially hydrophobic acid in the secondphase substantially concurrently with emulsifying the second phase.

126. The process of embodiment 125, wherein the first organic phasecomprises the therapeutic agent and the first aqueous solution comprisesthe substantially hydrophobic acid.

127. The process of any one of embodiments 121-126, wherein thetherapeutic agent, when protonated, has a first pK_(a), thesubstantially hydrophobic acid has a second pK_(a), and the emulsionphase is quenched with an aqueous solution having a pH equal to a pK_(a)unit between the first pK_(a) and the second pK_(a).128. The process of embodiment 127, wherein the quenched phase has a pHequal to a pK_(a) unit between the first pK_(a) and the second pK_(a).129. The process of any one of embodiments 121-128, wherein thetherapeutic agent, when protonated, has a first pK_(a), thesubstantially hydrophobic acid has a second pK_(a), and the firstaqueous solution has a pH equal to a pK_(a) unit between the firstpK_(a), and the second pK_(a).130. The process of any one of embodiments 127-129, wherein the pH isequal to a pK_(a) unit that is about equidistant between the firstpK_(a) and the second pK_(a).131. The process of any one of embodiments 121-130, wherein thetherapeutic agent is1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodiments,and are not intended to limit the invention in any way.

Example 1—Preparation of Formulation A with Therapeutic Agent

-   -   (a) Preparation of organic phase stock: Benzyl alcohol (8932.5        mg) was dissolved in 67.5 mg of RODI (reverse osmosis deionized)        water with mixing. The therapeutic agent,        1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,        (150 mg) was added to the solution, and then it was sonicated        until the drug dissolved. PLA-PEG-GL (19.2 mg) and PLA-PEG in a        ratio of 16 mol/5 mol (830.8 mg) was added thereto and vortexed        until dissolved.    -   (b) Preparation of aqueous phase stock: Sodium Cholate (2.75 g)        was dissolved in RODI water (955.5 g) on a stir plate. Benzyl        alcohol (40 g) was added to the sodium cholate/water solution        and the mixture was stirred on a stir plate until dissolved.    -   (c) Formation of emulsion: The weight ratio of aqueous phase to        organic phase was 5:1. The organic phase, which weighed 10 g,        was poured into 50 g of the aqueous phase that was cooled in ice        water bath, and the mixture homogenized using a hand homogenizer        for 15 seconds. The coarse emulsion was fed through a high        pressure homogenizer with pressure set at 10485 psi on gauge for        1 pass to form a nanoemulsion (fine emulsion).    -   (d) Formation of nanoparticles: The nanoemulsion was poured into        600 g of cold RODI water (less than 2° C.) while stirring on a        stir plate to form a quenched phase. (The weight ratio of quench        to emulsion is 10:1). To the quenched phase was added 64.3 grams        of a solution of polysorbate 80 (350 grams dissolved in 650 g        RODI water) with mixing.    -   (e) Concentration of nanoparticles through tangential flow        filtration (TFF): The quenched phase was concentrated using TFF        with 300 kDa Pall cassette (2 membranes) to form a nanoparticle        concentrate of approximately 200 mL. The nanoparticle        concentrate was diafiltered with approximately 20 diavolumes of        cold RODI water at less than 2° C. The volume of the diafiltered        nanoparticle concentrate was reduced to minimal volume.

Thus, this formulation contained1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,and the polymers PLA-PEG (in a 16:5 molar ratio) and PLA-PEG-GL in aweight ratio of PLA-PEG to PLA-PEG-GL of about 43:1 and a weight ratioof the therapeutic agent,1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,to polymers of 15:85. No counterion or hydrophobic acid was present inthis formulation. The particle size of a nanoparticle so formed asdescribed herein above was about 116 nm.

Example 2—Preparation of Formulation B with Therapeutic Agent

-   -   (a) Preparation of organic phase stock: Oleic acid (900 mg),        trifluoroacetic acid (TFA) (273 mg) was dissolved in benzyl        alcohol (8827 mg). The therapeutic agent,        1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea        (120 mg) was mixed with the oleic acid/TFA/benzyl alcohol        solution and heated to 80° C. for 10 minutes to dissolve the        therapeutic agent therein. Once the        1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea        was dissolved, the solution was allowed to cool to room        temperature. This solution was thoroughly mixed with a polymer        solution of PLA-PEG in a ratio of 16 moles/5 moles (860 mg),        PLA-PEG-GL (18.9 mg) and ethyl acetate (4549 mg) to form a        solution.    -   (b) Preparation of aqueous phase stock: Sodium Cholate (4.5 g)        was dissolved in RODI water (955.5 g) on a stir plate. Benzyl        alcohol (40 g) was added to the sodium cholate/water solution        and the mixture was stirred on a stir plate until dissolved.    -   (c) Formation of emulsion: The weight ratio of aqueous phase to        organic phase was 5:1. The organic phase was poured into 33.4 g        of the aqueous phase that was cooled in ice water bath, and the        mixture homogenized using a hand homogenizer for 15 seconds. The        coarse emulsion was fed through a high pressure homogenizer with        pressure set at 10485 psi on gauge for 1 pass to form a        nanoemulsion (fine emulsion).    -   (d) Formation of nanoparticles: The nanoemulsion was poured into        401.2 g of cold RODI water (less than 2° C.) while stirring on a        stir plate to form a quenched phase. To the quenched phase was        added 51.4 grams of a solution of polysorbate 80 (350 g        dissolved in 650 g RODI water) with mixing.    -   (e) Concentration of nanoparticles through tangential flow        filtration (TFF): The quenched phase was concentrated using TFF        with 300 kDa Pall cassette (2 membranes) to form a nanoparticle        concentrate of approximately 200 mL. The nanoparticle        concentrate was diafiltered with approximately 20 diavolumes of        cold RODI water at less than 2° C. The volume of the diafiltered        nanoparticle concentrate was reduced to minimal volume.

Thus, this formulation contained1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand the polymers PLA-PEG (in a 16:5 molar ratio) and PLA-PEG-GL in aweight ratio of PLA-PEG to PLA-PEG-GL of about 46:1 and a weight ratioof the therapeutic agent,1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,to polymers of 12:88. It contained about 5.7% by weight1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand about 9% by weight oleic acid in a 3% trifluoroacetic acid. Theparticle size of a nanoparticle so formed as described herein above wasabout 74 nm.

Example 3—Preparation of Formulation C with Therapeutic Agent

-   -   (a) Preparation of organic phase stock: Trifluoroacetic acid        (1600 mg), benzyl alcohol (8827 mg), and RODI water (1500 mg)        were mixed together and, if necessary, heated to form a        solution. To this solution was added the therapeutic agent,        1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea        (1468.8 mg), and the resulting mixture was sonicated to form a        solution. Once the therapeutic agent was dissolved, the solution        was allowed to cool to room temperature. This solution was added        to a solution of pamoic acid (136.5 mg) and DMSO (331.2 mg).        This solution was thoroughly mixed with a polymer solution of        PLA-PEG in a ratio of 16 mol/5 mol (643.5 mg), PLA-PEG-GL (14.5        mg) and ethyl acetate (7200 mg).    -   (b) Preparation of aqueous phase stock: A surfactant, Brij S 100        (polyoxyethylene (100) stearyl ether) (200 mg), was dissolved in        benzyl alcohol (40.0 g) with stirring, and cold RODI water        (959.8 g) was added thereto and mixed on ice until the solution        clears. The aqueous phase stock was cooled to less than 2° C.        with stirring.    -   (c) Formation of emulsion: The weight ratio of aqueous phase to        organic phase was 5:1. The organic phase was poured into 50.07 g        of the aqueous phase that was cooled in ice water bath, and the        mixture homogenized using a hand homogenizer for 15 seconds. The        coarse emulsion was fed through a high pressure homogenizer with        pressure set at 10485 psi on gauge for 1 pass to form a        nanoemulsion (fine emulsion).    -   (d) Formation of nanoparticles: The nanoemulsion was poured into        a quench solution of cold RODI water (1000 g) that was chilled        to less than 2° C. and was stirred on a stir plate. To the        quenched solution was added to a chilled solution (less than 2°        C.) of polysorbate 80 (350 g) dissolved in RODI water (650 g)        with mixing.    -   (e) Concentration of nanoparticles through tangential flow        filtration (TFF): The quenched phase was concentrated using TFF        with 300 kDa Pall cassette (2 membranes) to form a nanoparticle        concentrate of approximately 200 mL. The nanoparticle        concentrate was diafiltered with approximately 20 diavolumes of        cold RODI water at less than 2° C. The volume of the diafiltered        nanoparticle concentrate was reduced to minimal volume.

Thus, this formulation contained1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,and the polymers PLA-PEG (in a 16:5 molar ratio) and PLA-PEG-GL in aweight ratio of PLA-PEG to PLA-PEG-GL of about 44:1 and a weight ratioof the therapeutic agent,1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,to polymers of 22:64. It contained about 60% by weight of pamoic acid to1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea.Thus, the formulation contained about 5% by weight1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand a 3.2% by weight pamoic acid. The particle size of a nanoparticle soformed as described herein above was about 92 nm.

Example 4: Formulation D with Therapeutic Agent

-   -   (a) Preparation of organic stock solution: A 7 heated wt %        xinafoic acid solution in benzyl alcohol was combined with        PLA-PEG in a mole ratio of 16:5 with ethyl acetate was vortexed        until dissolved. The therapeutic agent,        1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,        was added thereto to make a final concentration of 15% by        weight.    -   (b) Preparation of aqueous phase stock: Sodium cholate (2.75 g)        was dissolved in RODI water (955.5 g) with stirring. Benzyl        alcohol (40 g) was added to the aqueous sodium cholate solution        and the mixture was stirred until dissolved.    -   (c) Formation of emulsion: The weight ratio of aqueous phase to        organic phase was 5:1. The organic phase was poured into the        aqueous phase which was cooled in ice water bath, and the        mixture was homogenized using a hand homogenizer for 15 seconds.        The coarse emulsion was fed through a high pressure homogenizer        with pressure set at 10485 psi on gauge for 1 pass to form a        nanoemulsion (fine emulsion).    -   (d) Formation of nanoparticles: The nanoemulsion was poured into        a quench buffer solution consisting of anhydrous citric acid        (19.2 g) in cold RODI water (1000 g) that was chilled to less        than 2° C., and brought to pH 4.5 with 10 N sodium hydroxide,        and the resulting solution stirred on a stir plate. To the        quenched solution was added a chilled solution (less than 2° C.)        of polysorbate 80 (350 g) dissolved in RODI water (650 g) with        mixing.    -   (e) The nanoparticles were concentrated through tangential flow        filtration on accordance with the procedure of Example 1.

Thus, this formulation contained the counterion xinafoic acid. Itcontained1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea.The particle size of a nanoparticle so formed as described herein abovewas about 109 nm.

Comparative Example 1 Control Solution

-   -   (a) Preparation of organic stock solution: A 7.5 wt % benzyl        alcohol solution, prepared by dissolving benzyl alcohol in RODI        water, was combined with PLA-PEG in a mixture having a mole        ratio of 16:5 with ethyl acetate and vortexed until dissolved.    -   (b) Preparation of aqueous phase stock: Sodium cholate (2.75 g)        was dissolved in RODI water (955.5 g) with stirring. Benzyl        alcohol (40 g) was added to the sodium cholate/water solution        and the mixture was stirred until dissolved.    -   (c) Formation of emulsion: The weight ratio of aqueous phase to        organic phase was 5:1. The organic phase was poured into the        aqueous phase that was cooled in ice water bath, and the mixture        homogenized using a hand homogenizer for 15 seconds. The coarse        emulsion was fed through a high pressure homogenizer with        pressure set at 10485 psi on gauge for 1 pass to form a        nanoemulsion (fine emulsion).    -   (d) Formation of nanoparticles: The nanoemulsion was poured into        600 g of cold RODI water (less than 2° C.) while stirring on a        stir plate to form a quenched phase. (The weight ratio of quench        to emulsion is 10:1). To the quenched phase was added 64.3 g of        a solution of polysorbate 80 (350 g dissolved in 650 g RODI        water) with mixing.    -   (e) Concentration of nanoparticles through tangential flow        filtration (TFF): The quenched phase was concentrated using TFF        with 300 kDa Pall cassette (2 membranes) to form a nanoparticle        concentrate of approximately 200 mL. The nanoparticle        concentrate was diafiltered with approximately 20 diavolumes of        cold RODI water at less than 2° C. The volume of the diafiltered        nanoparticle concentrate was reduced to minimal volume.

Comparative Example 2 Formulation B

-   -   (a) Preparation of organic phase stock: Oleic acid (900 mg),        trifluoroacetic acid (TFA) (273 mg) was dissolved in benzyl        alcohol (8827 mg). The therapeutic agent,        1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea        (120 mg) was mixed with the oleic acid/TFA/benzyl alcohol        solution and heated to 80° C. for 10 minutes to dissolve the        therapeutic agent therein. Once the        1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea        was dissolved, the solution was allowed to cool to room        temperature. This solution was thoroughly mixed with a polymer        solution of PLA-PEG in a ratio of 16 moles/5 moles (860 mg),        PLA-PEG-GL (18.9 mg) and ethyl acetate (4549 mg) to form a        solution.    -   (b) Preparation of aqueous phase stock: Sodium Cholate (4.5 g)        was dissolved in RODI water (955.5 g) on a stir plate. Benzyl        alcohol (40 g) was added to the sodium cholate/water solution        and the mixture was stirred on a stir plate until dissolved.    -   (c) Formation of emulsion: The weight ratio of aqueous phase to        organic phase was 5:1. The organic phase was poured into 33.4 g        of the aqueous phase that was cooled in ice water bath, and the        mixture homogenized using a hand homogenizer for 15 seconds. The        coarse emulsion was fed through a high pressure homogenizer with        pressure set at 10485 psi on gauge for 1 pass to form a        nanoemulsion (fine emulsion).    -   (d) Formation of nanoparticles: The nanoemulsion was poured into        401.2 g of cold RODI water (less than 2° C.) while stirring on a        stir plate to form a quenched phase. To the quenched phase was        added 51.4 grams of a solution of polysorbate 80 (350 g        dissolved in 650 g RODI water) with mixing.    -   (e) Concentration of nanoparticles through tangential flow        filtration (TFF): The quenched phase was concentrated using TFF        with 300 kDa Pall cassette (2 membranes) to form a nanoparticle        concentrate of approximately 200 mL. The nanoparticle        concentrate was diafiltered with approximately 20 diavolumes of        cold RODI water at less than 2° C. The volume of the diafiltered        nanoparticle concentrate was reduced to minimal volume.

Thus, this formulation contained1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand the polymers PLA-PEG (in an about 16:5 molar ratio) in a weightratio of the therapeutic agent to polymers of about 1:14.7. It containedabout 6.0% by weight1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,about 5.4% cholic acid, and about 1.1% by weight oleic acid.

Example 5: Formulation E with Therapeutic Agent

The procedure of Example 1 was repeated except that there was noPLA-PEG-GL polymer present. The PLA-PEG-GL polymer was replaced with19.2 mg of PLA-PEG in a ratio of 16 mol/5 mol so that the total amountof PLA-PEG present was 850 mg.

Example 6: Formulation F with Therapeutic Agent

The procedure of Example 2 was repeated except that there was noPLA-PEG-GL polymer present. The PLA-PEG-GL polymer was replaced with 20mg of PLA-PEG in a ratio of 16 mol/5 mol so that the total amount ofPLA-PEG present was 860 mg.

Example 7: Formulation F with Therapeutic Agent

The procedure of Example 3 was repeated except that there was noPLA-PEG-GL polymer present. The PLA-PEG-GL polymer was replaced with14.5 mg of PLA-PEG in a ratio of 16 mol/5 mol so that the total amountof PLA-PEG present was 658 mg.

Example 8: Release Profile of Formulation

Each formulation was prepared at a scale sufficient to deliver 200 mgtherapeutic agent,1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea,at a concentration of >2.5 mg/mL (FORMULATION A=25 g, FORMULATION B=20g, FORMULATION C=10 g). Nanoparticle suspensions were prepared with 30wt % sucrose and vialed in >11 mg of therapeutic agent aliquots. Table 1summarizes the attributes of nanoparticles prepared for this study.

TABLE 1 Summary of FORMULATION A, B, C Therapeutic Agent Particle SizeAPI Released Formulation Lot Number Loading (nm) at 24 hr A 237-46 4%130 60% B 237-45 5% 95 22% C 237-44 16% 100 2%

The three batches satisfied particle size and therapeutic agent releasecriteria (90-150 nm, <50% therapeutic agent released at t>2 h). With theexception of the nanoparticles of FORMULATION A, the batches also metthe therapeutic agent loading criteria of >5%. Historically, thetherapeutic agent loading for FORMULATION A has been on the lower limitor below the target loading threshold, so this result was notunexpected.

FIG. 3 shows the in vitro release curves for each batch. The in vitrorelease method used determines release profiles from these nanoparticlesat 37° C. conditions using the centrifugal system. Samples werecentrifuged at 264,000×g for 30 minutes and the supernatant was assayedfor therapeutic agent concentration. Cumulative release percentage wasdetermined by comparing the supernatant concentration with the totaltherapeutic agent concentration prior to centrifugation.

The in vitro release profile in FIG. 3 shows that the rate of therelease of therapeutic agent was quantifiably distinct for each of theformulations. FIG. 4 depicts the pharmacokinetics of the therapeuticagent nanoparticles in Wistar Han Rats.

The protocol was as follows: Male Wistar Han rats (approximately sixweeks in age; n=4/group) with indwelling jugular vein cannulae weredosed intravenously with a 1 mg/kg bolus of Formulation A, B, and Cnanoparticles or Formulation A, B, and C nanoparticles diluted in 0.9%saline. At various times after dosing, serial blood collections weremade from the jugular vein cannulae and plasma concentrations oftherapeutic agent were quantitated by LC-MS/MS. FIG. 4(a) shows thepharmacokinetics of nanoparticles vs. free therapeutic agent, while (b)shows the same data with free therapeutic agent omitted.

FIG. 4(a) indicates that all three formulations A, B, and C, that weretested exhibited substantially increased retention times in the bloodstream over the free API. This corresponds to increased values of AUCand t_(1/2), summarized in Table 2 (TA=therapeutic agent).

TABLE 2 Summary of AUC_(all) and t_(1/2) data for FORMULATION A, B & Cnanoparticles tested. Formulation 7% Xinafoic Formulation FormulationParameter TA A 60% EA Acid C B AUC_(all) (hr · ng/mL) 919.5 312,972485,188 653,749 601,768 550,539 t_(1/2) (hr) 13.2 17.7 23.8 25.1 23.823.7

Example 9: Release Profile for Formulation C

FORMULATION C was again prepared as in Example 3 using the traditionalbatch process at the 2 g and 5 g scale. An additional 2 g batch ofFormulation C was prepared using a 50 mM citric acid buffer titrated topH 4.5 with sodium hydroxide to promote potential ion pairing. This pHwas chosen because it was in between the pK_(a), of pamoic acid (˜2.5)and the first pK_(a) of1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea(˜6.7). Table 3 summarizes the particle attributes for these small-scalebatches:

TABLE 3 Effect of using pH 4.5 50 mM citric acid buffer for the quenchmedium in Formulation C. Therapeutic Agent Batch Lot Number Loading Size(nm) 2 g RODI Quench 237-34-1 4.48% 101 5 g RODI Quench 237-34-2 2.43%98 2 g Buffered Quench 237-34-3 13.18% 92

The increased emulsion processing time from a 2 g batch to a 5 g batchresulted in a substantial drop in the loading of the therapeutic agent.However, it was shown that using a pH 4.5 buffered quench resulted innearly a three-fold increase in loading of the therapeutic agent,1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea.

The in vitro release method was used to determine the release profilesfrom these nanoparticles at 37° C. conditions using the centrifugalsystem. Samples were centrifuged at 264,000×g for 30 minutes and thesupernatant was assayed for therapeutic agent concentration. Cumulativerelease percentage was determined by comparing the supernatantconcentration with the total therapeutic agent concentration prior tocentrifugation. FIG. 5 shows the in vitro release profile was unaffectedby the use of a buffered quench.

Example 10: Determination of Particle Attributes for Formulation C

Two 10 g batches of Formulation C were prepared with 100 mM citric acidbuffer quench titrated to pH 4.5, each by pooling five 2 g batches toavoid the effects of processing time on drug loading of the therapeuticagent,1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea.Table 4 summarizes the particle attributes for these batches.

TABLE 4 Particle attributes for FORMULATION C batches utilizing pH 4.5citric acid buffer quench. Target Organic Particle Lot API PhaseFormulation C Size Number Description Loading Solids Loading (nm)Surfactant 237-34-3 8 wt % TFA 7.5 wt water in 22% 10% 13.18% 92 Brij,0.02 wt % BA, 1:1 pamoic to therapeutic agent, 20:80 (BA + DMSO):EA, 50mM citric acid quench, pH 4.5 237-36 8 wt % TFA 7.5 wt % water in 22%10% 18.25% 100 Brij, 0.02 wt % BA, 1:1 pamoic to therapeutic agent,20:80 (BA + DMSO):EA, 100 mM citric acid quench, pH 4.5 237-44 8 wt %TFA 7.5 wt % water in 22% 10% 16.30% 100 Brij, 0.02 wt % BA, 1:1 pamoicto therapeutic agent, 20:80 (BA + DMSO):EA, 100 mM citric acid quench,pH 4.5, GL Targeted BA = benzyl alcohol EA = ethyl alcohol

The in vitro release profiles were conducted as follows; the in vitrorelease method was used to determine the release profiles from thesenanoparticles at 37° C. conditions using the centrifugal system. Samplesare centrifuged at 264,000×g for 30 minutes and the supernatant wasassayed for therapeutic agent concentration. Cumulative releasepercentage was determined by comparing the supernatant concentrationwith the total therapeutic agent concentration prior to centrifugation.The results are shown in FIG. 6.

From the investigations, it was ascertained that maximum therapeuticagent loading in the formulation C was achieved at pH 4.5. Withoutwishing to be bound, it is believed that this may be attributed to thefact that ion pairing between the therapeutic agent and the counter ionis encouraged when the pH of the solution is below the pK_(a) of theprotonated therapeutic agent drug and above the pK_(a) of the acidicmolecule (pamoic acid). This effect is believed to be theoreticallymaximized when the largest fraction of both species are in their ionizedstate.

Example 11: MDAMB361 Xenograft Scheduling Study1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaNanoparticles Q4D Versus Q8D

Female SCID/bg mice at age around 6 weeks were obtained from CharlesRiver Laboratories (Wilmington, Mass.). Animals were maintained underclean room conditions in sterile filter top cages with Alpha-Dri beddingand housed on HEPA-filtered ventilated racks. Animals received sterilerodent chow and water ad libitum. All of the procedures were conductedin accordance with the Institute for Laboratory Animal Research Guidefor the Care and Use of Laboratory Animals and with Pfizer Animal Careand Use Committee guidelines.

Three to four days prior to tumor cell inoculation, the animals wereimplanted with a 0.36 mg, 60-d release 17β-estradiol pellet (InnovativeResearch of America). The MDAMB-361 cells which were harvested at 80-90%confluence and viability above 80-90% (NS) were supplemented with 50%Matrigel (BD Biosciences, San Jose Calif.) to facilitate tumor take.Cells (5×106 in 200 μL) were implanted subcutaneously (S.C.) into thehind flank region of the mouse and allowed to grow to the designatedsize prior to the administration of compound for each experiment. Tumorsize was determined by measurement with an electronic calipers and tumorvolume was calculated as the product of its length×width²×0.5. Whentumor volumes reached an average of 250 mm³, mice were randomized fortreatment groups including vehicle control group with intravenous (i.v.)injections of the corresponding drug at 10 mL/kg volume on an everyfour-day (Q4D) or eight-day (Q8D) schedule. Animals were treated with 5or 10 mg/kg1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor 25 mg/kg of the Formulation B nanoparticle at each injection.

FIG. 7 shows that Formulation B nanoparticles dosed every 8 days hassimilar efficacy as once every 4 days and that Formulation Bnanoparticles may afford a 2 week dosing frequency in the clinic.

Example 12: MDAMB361 Tumor Growth Inhibition and Tumor Growth DelayStudy

Female SCID/bg mice at age around 6 weeks were obtained from CharlesRiver Laboratories (Wilmington, Mass.). Animals were maintained underclean room conditions in sterile filter top cages with Alpha-Dri beddingand housed on HEPA-filtered ventilated racks. Animals received sterilerodent chow and water ad libitum. All of the procedures were conductedin accordance with the Institute for Laboratory Animal Research Guidefor the Care and Use of Laboratory Animals and with Pfizer Animal Careand Use Committee guidelines.

Three to four days prior to tumor cell inoculation, the animals wereimplanted with a 0.36 mg, 60-d release 17β-estradiol pellet (InnovativeResearch of America). The MDAMB-361 cells which were harvested at 80-90%confluence and viability above 80-90% (NS) were supplemented with 50%Matrigel (BD Biosciences, San Jose Calif.) to facilitate tumor take.Cells (5×106 in 200 μL) were implanted subcutaneously (S.C.) into thehind flank region of the mouse and allowed to grow to the designatedsize prior to the administration of compound for each experiment. Tumorsize was determined by measurement with an electronic calipers and tumorvolume was calculated as the product of its length×width²×0.5. Whentumor volumes reached an average of 250 mm³, mice were randomized fortreatment groups including vehicle control group with intravenous (i.v.)injections of the corresponding drug at 10 mL/kg volume on an everyfour-day (Q4D) schedule for 4 doses. Post the 4th dose, animals werefurther monitored for tumor growth delay. Animals were treated with 10mg/kg1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea;2, 10, or 25 mg/kg of the Formulation A or B nanoparticles; or 10 or 25mg/kg of the Formulation C nanoparticles at each injection.

FIGS. 8A, 8B, and 8C show that Formulation B nanoparticles andFormulation C nanoparticles inhibit tumor growth with improved efficacyversus1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea(naked API) and Formulation A nanoparticles.

Example 13: WM266-4 Model Tumor Growth Inhibition Study

Female nu/nu mice at age around 8 weeks were obtained from Charles RiverLaboratories (Wilmington, Mass.). Animals were maintained under cleanroom conditions in sterile filter top cages with Alpha-Dri bedding andhoused on HEPA-filtered ventilated racks. Animals received sterilerodent chow and water ad libitum. All of the procedures were conductedin accordance with the Institute for Laboratory Animal Research Guidefor the Care and Use of Laboratory Animals and with Pfizer Animal Careand Use Committee guidelines.

The WM266-4 cells which were harvested at 80-90% confluence andviability above 80-90% (NS) were supplemented with 50% Matrigel (BDBiosciences, San Jose Calif.) to facilitate tumor take. Cells (2×10⁶ in200 μL) were implanted subcutaneously (S.C.) into the hind flank regionof the mouse and allowed to grow to the designated size prior to theadministration of compound for each experiment. Tumor size wasdetermined by measurement with an electronic calipers and tumor volumewas calculated as the product of its length×width²×0.5. When tumorvolumes reached an average of 400 mm³, mice were randomized fortreatment groups including vehicle control group with oral daily (QD) ofPF-0192513-00-0004 (PD-901) and/or intravenous (i.v.) injections of thenanoparticle drugs, B and C at 10 mL/kg volume on an every four-day(Q4D) schedule for 4 doses. Dosing and drug are described in the figurelegends. Animals were treated with 10 mg/kg1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor 10, 25, or 50 mg/kg of the Formulation B or C nanoparticles at eachinjection.

FIG. 9 illustrates that Formulation C nanoparticles produce greatertolerability and efficacy than Formulation B nanoparticles or1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea(naked API).

Example 14: In Vivo Target Modulation Studies with Nanoparticles

In vivo target modulation studies were conducted to determine theeffects of treatment with the Formulation A, B, and C nanoparticles onthe phosphorylation of S6 on S235/S236 and AKT on S473 and T308 byELISA. Resected fresh tumors were ground into fine powder using metalMartor and granite pestle under liquid nitrogen. The tumor powder wasstored at −80° C. until preparation of tumor lysates for ELISA assay.Briefly, an aliquot (50 mg) of tumor powder was put into pre-cold 2 mLglass martor tube, 500 μl cold lysis buffer [20 mM Tris-HCl (pH 7.5),150 mM NaCl, 1.0 mM Na₂EDTA, 1 mM EGTA, 1% NP-40, 1% sodiumdeoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mMNa₃VO₄, 1 μg/ml leupeptin, 1 mM PMSF, 1× protease/phosphatase inhibitorcocktail] was added, the tube was embedded in wet ice, and samples werehomogenized at speed 6 for 30 seconds by using a tissue homogenizer.Samples were collected and snap frozen on dry ice and thawed on wet ice.Repeated freeze-thaw cycle, then centrifuged samples in a coldrefrigerated Eppendorf centrifuge at 13,000 rpm for 10 minutes.Supernatant was collected and centrifuged again. The total andphosphoAKT (S473 and T308) and the total and phosphoS6 protein levels intumor lysates were determined by ELISA. The extent of phosphorylation intumors resected from treated animals was compared with that in tumorsresected from vehicle-treated animals at the same time point.

FIGS. 8A, 8B, and 8C show that Formulation B nanoparticles andFormulation C nanoparticles inhibit pS6 with improved efficacy versus1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea(naked API) and Formulation A nanoparticles, and demonstrate persistenttarget modulation observed up to day 7 post-dose.

Example 15: Analysis of Glucose and Insulin Levels after NanoparticleTreatment

Glucose:

In mouse or rat studies, approximately 100 μL plasma (EDTA asanticoagulant) was used for assessment of glucose content based on anenzymatic assay as published by Slein (Bergmeyer H U, ed. Slein M W.Methods of Enzymatic Analysis. New York, N.Y.: Academic Press;1974:1196-1201.), using hexokinase and glucose-6-phosphate dehydrogenaseenzymes. Plasma glucose was measured with Advia@120 Glucose Hexokinase_3(GLUH_3) system with Automated Hematology Analyzer (Siemens HealthcareDiagnostics Inc., Tarrytown, N.Y.). The Advia Chemistry GlucoseHexokinase_3 (GLUH_3) assay used a two-component reagent. Plasma samplewas added to Reagent 1, which contained the buffer, ATP, and NAD.Absorbance readings of the sample in Reagent 1 were taken and used tocorrect for interfering substances in the sample. Reagent 2 (the buffer,ATP, NAD, Hexokinase, and G6PD) was added, which initiated theconversion of glucose and the development of absorbance at 340/410 nm.The difference between the absorbance in Reagent 1 and Reagent 2 wasproportional to the glucose concentration.

Insulin:

In mouse or rat studies, approximately 20 μL plasma (EDTA asanticoagulant) was used for assessment of insulin content. The insulinassay was a Sandwich ELISA based using Rat/Mouse Insulin ELISA Kitacquired from EMD Millipore Corporation (St. Charles, Mo.). The assayprocedure was as follows: 1) capture of insulin molecules from plasmasamples to the wells of a microtiter plate coated by pre-titered amountof a monoclonal mouse anti-rat insulin antibodies and the binding ofbiotinylated polyclonal antibodies to the captured insulin, 2) wash awayunbound materials from samples, 3) bind horseradish peroxidase to theimmobilized biotinylated antibodies, 4) wash away free enzymeconjugates, and 5) quantify immobilized antibody-enzyme conjugates bymonitoring horseradish peroxidase activities in the presence of thesubstrate 3,3′,5,5′-tetramethylbenzidine. The enzyme activity wasmeasured spectrophotometrically by the increased absorbance at 450 nm,which was directly proportional to the amount of captured insulin in theplasma sample. The plasma insulin concentration was calculated byinterpolation from a reference curve generated in the same assay withreference standards of known concentrations of rat or mouse insulin.

FIG. 10 illustrates that Formulation B and C nanoparticles may have animproved safety profile over the1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea(naked API).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A therapeutic nanoparticle comprising: atherapeutic agent selected from the group consisting of1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaand pharmaceutically acceptable salt thereof; a polymer selected fromthe group consisting of diblock poly(lactic) acid-poly(ethylene)glycol(PLA-PEG) copolymer, diblock poly(lactic acid-co-glycolicacid)-poly(ethylene)glycol (PLGA-PEG) copolymer, and combinationsthereof; and a substantially hydrophobic acid selected from the groupconsisting of dioctyl sulfosuccinic acid, 1-hydroxy-2-naphthoic acid,dodecyl sulfuric acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, pamoic acid, undecanoic acid, andcombinations thereof; wherein the substantially hydrophobic acid and thetherapeutic agent form a hydrophobic ion pair that is encapsulated inthe therapeutic nanoparticle; and wherein the hydrophobic ion pair isdispersed throughout a polymeric matrix comprising the polymer, whereinPLA-PEG-GL is additionally present, wherein targeting ligand GL has thefollowing structure:


2. The therapeutic nanoparticle according to claim 1, wherein the pK_(a)of the protonated therapeutic agent is at least about 1.0 pK_(a) unitsgreater than the pK_(a) of the substantially hydrophobic acid.
 3. Thetherapeutic nanoparticle according to claim 1 comprising about 0.2 toabout 20 weight percent of the therapeutic agent.
 4. The therapeuticnanoparticle according to claim 1 comprising about 50 to about 99.75weight percent of the polymer, and wherein the therapeutic nanoparticlecomprises about 10 to about 30 weight percent poly(ethylene)glycol. 5.The therapeutic nanoparticle according to claim 1 comprising about 0.05to about 30 weight percent of the substantially hydrophobic acid.
 6. Thetherapeutic nanoparticle according to claim 1 comprising:1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea;and PLA-PEG (in a 16:5 molar ratio) in a weight ratio of about 1:71-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea:PLA-PEG.7. The therapeutic nanoparticle according to claim 1 comprising:1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea;and PLA-PEG (in a 16:5 molar ratio) in a weight ratio of about 1:51-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea:PLA-PEG.8. The therapeutic nanoparticle according to claim 1 comprising:1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea;and PLA-PEG (in a 16:5 molar ratio) in a weight ratio of about 1:141-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea:PLA-PEG.9. The therapeutic nanoparticle according to claim 1, wherein the molarratio of the substantially hydrophobic acid to the therapeutic agentranges from about 0.25:1 to about 2:1.
 10. The therapeutic nanoparticleaccording to claim 1, wherein the molar ratio of the substantiallyhydrophobic acid to the therapeutic agent is about 0.25:1 to about 1:1.11. The therapeutic nanoparticle according to claim 1, wherein thehydrophobic acid is pamoic acid.
 12. The therapeutic nanoparticleaccording to claim 1 comprising about 0.2 to about 30 weight percent ofthe PLA-PEG-GL.
 13. The therapeutic nanoparticle according to claim 1,wherein the substantially hydrophobic acid is a mixture of two or moresubstantially hydrophobic acids.
 14. The therapeutic nanoparticle ofclaim 1 prepared by the process comprising the steps of: emulsificationof a first organic phase comprising a first polymer, a therapeuticagent, and a substantially hydrophobic acid, thereby forming an emulsionphase; quenching of the emulsion phase thereby forming a quenched phase;and filtration of the quenched phase to recover the therapeuticnanoparticles, wherein the therapeutic agent is1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]ureaor a pharmaceutically acceptable salt thereof, wherein the substantiallyhydrophobic acid and the therapeutic agent form a hydrophobic ion pairin the therapeutic nanoparticle.
 15. The therapeutic nanoparticleaccording to claim 1, further comprising a solubilizer.
 16. Apharmaceutical composition comprising a therapeutic nanoparticleaccording to claim 1 and a pharmaceutically acceptable excipient. 17.The pharmaceutical composition of claim 16 comprising a plurality oftherapeutic nanoparticles.